Back to EveryPatent.com
| United States Patent |
6,160,090
|
|
Schlessinger
, et al.
|
December 12, 2000
|
Receptor protein tyrosine phosphatases
Abstract
A novel receptor-type protein tyrosine phosphatase-.beta. (RPTP.beta.)
protein or glycoprotein is disclosed. This protein is naturally expressed
in the brain and in neural cell lines.
| Inventors:
|
Schlessinger; Joseph (New York, NY);
Barnea; Gilad (New York, NY);
Grumet; Martin Hyman (New York, NY);
Margolis; Richard U. (New York, NY)
|
| Assignee:
|
New York University (New York, NY)
|
| Appl. No.:
|
081929 |
| Filed:
|
June 23, 1993 |
| Current U.S. Class: |
530/350; 530/395 |
| Intern'l Class: |
C07K 001/00; C07K 014/00; C07K 017/00 |
| Field of Search: |
530/350,395
435/69.1,183,7.1,196
|
References Cited
Other References
Kiang, W.-L. et al., 1981, J. Biol. Chem. 256:10529-10537.
Hoffman, S. et al., 1982, J. Biol. Chem. 257:7720-7729.
Hoffman, S. and Edelman., G.M., 1983, Proc. Natl. Acad. Sci. USA
80:5762-5766.
Grumet, M. et al., 1984, Proc. Natl. Sci. USA 81:267-271.
Thomas, M.L. et al., Cell 41:83-93.
Durst, M. et al., 1987, Proc. Natl. Acad. Sci USA 84:1070-1074.
Ralph, S.J. et al., 1987, EMBO J. 6:1251-1257.
Deutsch, H.F., 1987, Int. J. Biochem. 19:101-113.
Hunter, T., 1987, Cell 49:1-14.
Thomas, M.L. et al., 1987, Proc. Natl. Acad. Sci. USA 84:5360-5363.
Williams, A.F., 1987, Immunol. Today 8:298-303.
Honegger, A.M. et al., 197, Cell 51:199-209.
Doege, K.M. et al, 1987, J. Biol. Chem. 262:17757-17767.
Grumet, M. and Edelman, G.M., 1988, J. Cell Biol. 106:487-503.
Streuli, M. et al., 1988, J. Exp. Med. 168:1523-1530.
Ledbetter, J.A. et al., 1988, Proc. Natl. Acad. Sci. USA 85:8628-8632.
Margolis, B. et al., 1989, Cell 57:1101-1107.
Charbonneau, H. et al., 1989, Proc. Natl. Acad. Sci. USA 86:5252-5256.
Cool, D.E. et al., 1989, Proc. Natl. Acad. Sci. USA 86:5257-5261.
Morla, A.O. et al., 1989, Cell 58:193-203.
Hunter, T., 1989, Cell 58:1013-1016.
Streuli, M. et al., 1989, Proc. Natl. Acad. Sci. USA 86:8698-8702.
Guan, K. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1501-1505.
Krueger, N.X. et al.., 1990, EMBO J. 9:3241-3252.
Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212.
Hardie, D.G., 1990, Symp. Soc. Exp. Biol. 44:241-255.
Nurse, O., 1990, Nature 344:503-508.
Sap, J. et al., 1990, Proc. Natl. Acad. Sci. USA 87:6112-6116.
Kaplan, R. et al., 1990, Proc. Natl. Acad. Sci. USA 87:7000-7004.
Jirik, F.R. et al., 1990, FEBS Lett. 273:239-242.
Hunter T., 1991, Cell 64:249-270.
Fischer, E.H. et al., 1991 Science 253:401-406.
Cantley, L.C. et al., 1991, Cell 64:281-302.
Takeichi, M., 1991, Science 251:1451-1455.
Nada, S. et al., 1991, Nature 351:69-72.
LaForgia, S. et al., 1991, Proc. Natl. Acad. Sci. USA 88:5036-5040.
Cannizzano, L.A. et al., 1991, Cancer Res. 51:3818-3820.
Lombroso et al., 1991, Proc. Natl. Acad. Sci. USA 88:7242-7246.
Rauch, U. et al., 1991, J. Biol. Chem. 266:14785-14801.
Posada, J. and Cooper, J.A., 1992, Mol. Biol. Cell 3:583-592.
Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391.
Scott, J.D. and Soderling, T.R., 1992, Current Opinion in Neurobiology
2:289-295.
Erickson, H.P., 1989, Ann. Rev. Cell Biol. 5: 71-92.
Sawada et al., J. of Biol. Chem., vol. 268, pp. 12675-12681, 1993.
Krueger et al., PNAS, vol. 89, p. 7417, 1992.
Sambrook et al., Molecular Cloning, A Laboratory Manual, vol. 3, pp.
16.2-16.30, 1989.
|
Primary Examiner: Kunz; Gary L.
Assistant Examiner: Landsman; Robert S.
Attorney, Agent or Firm: Foley & Lardner
Parent Case Text
This is a Continuation-In-Part of Ser. No. 07/961,235, filed Oct. 15, 1992,
now abandoned, which is incorporated by reference herein in its entirety,
and of Ser. No. 08/015,973, filed Feb. 10, 1993, now U.S. Pat. 5,604,094,
which is a Continuation-In-Part of Ser. No. 07/654,188, filed Feb. 26,
1991, now abandoned, which is a Continuation-In-Part of Ser. No.
07/551,270, filed Jul. 11, 1990, now abandoned.
Claims
What is claimed is:
1. An isolated polypeptide comprising the amino acid sequence depicted in
SEQ ID NO:2.
2. An isolated polypeptide comprising one of the following amino acid
sequences depicted in SEQ ID NO:2: amino acid residues 1-21, 1-1636,
22-1636, 22-2308, 33-301, 1637-1662, 1663-2308, 1743-1984 or 2041-2274.
3. The isolated polypeptide of claim 1, wherein the polypeptide further
comprises a glycosaminoglycan chain.
4. The isolated polypeptide of claim 2, wherein the polypeptide further
comprises a glycosaminoglycan chain.
5. An isolated polypeptide comprising the amino acid sequence of SEQ ID
NO:2, but which lacks amino acid residues 755-1614 of SEQ ID NO:2.
6. The isolated polypeptide of claim 5, wherein the polypeptide further
comprises a glycosaminoglycan chain.
7. The isolated polypeptide of claim 2 comprising amino acid residues 1-21
of SEQ ID NO:2.
8. The isolated polypeptide of claim 2 comprising amino acid residues
1-1636 of SEQ ID NO:2.
9. The isolated polypeptide of claim 2 comprising amino acid residues
22-1636 of SEQ ID NO:2.
10. The isolated polypeptide of claim 2 comprising amino acid residues
22-2308 of SEQ ID NO:2.
11. The isolated polypeptide of claim 2 comprising amino acid residues
33-301 of SEQ ID NO:2.
12. The isolated polypeptide of claim 2 comprising amino acid residues
1637-1662 of SEQ ID NO:2.
13. The isolated polypeptide of claim 2 comprising amino acid residues
1663-2308 of SEQ ID NO:2.
14. The isolated polypeptide of claim 2 comprising amino acid residues
1742-1983 of SEQ ID NO:2.
15. The isolated polypeptide of claim 2 comprising amino acid residues
2040-2273 of SEQ ID NO:2.
Description
1. INTRODUCTION
The present invention relates to a new class of receptor protein tyrosine
phosphatase molecule, the ligands that bind this new class of receptor,
and the uses of such receptors and ligands. Specifically, the members of
this new class of receptor protein tyrosine phosphatase molecule are
proteoglycans and/or possess an extracellular carbonic anhydrase
structural domain. The characterization of one member of this new class,
RPTP.beta., is described in the working examples presented herein. As is
further demonstrated in the Working Examples presented below, the ligands
which bind the receptor protein tyrosine phosphatases of the invention may
be, for example, tenascin and/or members of the cell adhesion molecule
(CAM) family of extracellular molecules.
2. BACKGROUND OF THE INVENTION
2.1 PROTEIN PHOSPHORYLATION AND SIGNAL TRANSDUCTION
Cells rely, to a great extent, on extracellular molecules as a means by
which to receive stimuli from their immediate environment. These
extracellular signals are essential for the correct regulation of such
diverse cellular processes as differentiation, contractility, secretion,
cell division, cell migration, contact inhibition, and metabolism. The
extracellular molecules, which can include, for example, hormones, growth
factors, or neurotransmitters, act as ligands that bind specific cell
surface receptors. The binding of these ligands to their receptors
triggers a cascade of reactions that brings about both the amplification
of the original stimulus and the coordinate regulation of the separate
cellular processes mentioned above.
A central feature of this process, referred to as signal transduction (for
recent reviews, see Posada, J. and Cooper, J. A., 1992, Mol. Biol. Cell
3:583-592; Hardie, D. G., 1990, Symp. Soc. Exp. Biol. 44:241-255), is the
reversible phosphorylation of certain proteins. The phosphorylation or
dephosphorylation of amino acid residues triggers conformational changes
in regulated proteins that alter their biological properties. Proteins are
phosphorylated by protein kinases and are dephosphorylated by protein
phosphatases. Protein kinases and phosphatases are classified according to
the amino acid residues they act on, with one class being serine-threonine
kinases and phosphatases (reviewed in Scott, J. D. and Soderling, T. R.,
1992, 2:289-295), which act on serine and threonine residues, and the
other class being the tyrosine kinases and phosphatases (reviewed in
Fischer, E. H. et al., 1991 Science 253:401-406; Schlessinger, J. and
Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger, J.,
1990, Cell 61:203-212), which act on tyrosine residues. The protein
kinases and phosphatases may be further defined as being receptors, i.e.,
the enzymes are an integral part of a transmembrane, ligand-binding
molecule, or as non-receptors, meaning they respond to an extracellular
molecule indirectly by being acted upon by a ligand-bound receptor.
Phosphorylation is a dynamic process involving competing phosphorylation
and dephosphorylation reactions, and the level of phosphorylation at any
given instant reflects the relative activities, at that instant, of the
protein kinases and phosphatases that catalyze these reactions.
While the majority of protein phosphorylation occurs at serine and
threonine amino acid residues, phosphorylation at tyrosine residues also
occurs, and has begun to attract a great deal of interest since the
discovery that many oncogene products and growth factor receptors possess
intrinsic protein tyrosine kinase activity. The importance of protein
tyrosine phosphorylation in growth factor signal transduction, cell cycle
progression and neoplastic transformation is now well established
(Cantley, L. C. et al., 1991, Cell 64:281-302; Hunter T., 1991, Cell
64:249-270; Nurse, 1990, Nature 344:503-508; Schlessinger, J. and Ullrich,
A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger, J., 1990, Cell
61:203-212). Subversion of normal growth control pathways leading to
oncogenesis has been shown to be caused by activation or overexpression of
tyrosine kinases which constitute a large group of dominant oncogenic
proteins (reviewed in Hunter, T., 1991, Cell 64:249-270).
In addition, since the initial purification, sequencing and cloning of a
protein tyrosine phosphatase (Thomas, M. L. et al., 1985, Cell 41:83),
additional potential protein tyrosine phosphatases have been identified at
a rapid pace. (See, for example, Kaplan, R. et al., 1990, Proc. Natl.
Acad. Sci. USA 87:7000-7004; Krueger, N. X. et al., 1990, EMBO J.
9:3241-3252; Sap, J. et al., 1990, Proc. Natl. Acad. Sci. USA
87:6112-6116). Because the number of different protein tyrosine
phosphatases that have been identified is increasing steadily, speculation
has arisen that the protein tyrosine phosphatase family may be as large as
the protein tyrosine kinase family (Hunter, T., 1989, Cell 58:1013-1016).
With this increase in the reported cloning of protein tyrosine phosphatase
genes, the role that the regulation of dephosphorylation may have in the
control of cellular processes has also begun to receive more attention.
2.2 PROTEIN TYROSINE PHOSPHATASES
As mentioned above, protein tyrosine phosphatases (PTPases) can be
classified into two subgroups, the non-receptor and receptor classes. The
non-receptor class is composed of low molecular weight, cytosolic, soluble
proteins. All known non-receptor PTPases contain a single conserved
catalytic phosphatase domain of approximately 230 amino acid residues.
(See, for example, Charbonneau et al., 1989, Proc. Natl. Acad. Sci. USA
86:5252-5256; Cool et al., 1989, Proc. Natl. Acad. Sci. USA 86:5257-5261;
Guan et al., 1990, Proc. Natl. Acad. Sci. USA 87:1501-1502; Lombroso et
al., 1991, Proc. Natl. Acad. Sci. USA 88:7242-7246). Sequence analysis
reveals that about 40 of the amino acids of the catalytic domain are
highly conserved, and a very highly conserved segment of 11 amino acid
residues with the consensus sequence [I/V]HCXAGXXR[S/T]G [SEQ ID NO:1], is
now recognized to be a hallmark of the protein tyrosine phosphatase
catalytic domain.
The receptor class is made up of high molecular weight, receptor-linked
PTPases, termed RPTPases. Structurally resembling growth factor receptors,
RPTPases consist of an extracellular, putative ligand-binding domain, a
single transmembrane segment, and an intracellular catalytic domain
(reviewed in Fischer et al., 1991, Science 253:401-406). The intracellular
segments of almost all RPTPases are very similar. These intracellular
segments consist of two catalytic phosphatase domains of the type
described above, separated by an approximately 58 amino acid residue
segment. This two domain motif is usually located approximately 78 to 95
amino acid residues from the transmembrane segment and is followed by a
relatively short carboxy-terminal amino acid sequence. The only known
exception is the isoform HPTP.beta. (Krueger, N. X. et al., 1990, EMBO J.
9:3241), which contains only one catalytic phosphatase domain.
While the intracellular RPTPase segments are remarkably highly conserved,
the RPTPase extracellular domains are highly divergent. For example,
certain RPTPases possess a heavily glycosylated external domain and a
conserved cysteine-rich region (Thomas, M. L. et al., 1985, Cell 41:83;
Thomas, M. L. et al., 1987, Proc. Natl. Acad. Sci. USA 84:5360; Ralph, S.
J. et al., 1987, EMBO J. 6:1251-1257) while others contain immunoglobulin
G-like (Ig) domains linked to fibronectin type III repeats (Streuli, M. et
al., 1989, Proc. Natl. Acad. Sci. USA 86:8698; Streuli, M. et al., 1988,
J. Exp. Med. 168:1523). Still other RPTPases contains only multiple
fibronectin type III repeats (Krueger, N. X. et al., 1990, EMBO J.
9:3241), while certain RPTPases have smaller external domains that contain
several potential glycosylation sites (Jirik, F. R. et al., 1990, FEBS
Lett. 273:239). The ligands that regulate RPTPs have not been identified.
It has been speculated that circulating extracellular factors are unlikely
to bind to those receptors containing Ig and/or fibronectin Type III
repeats and that interaction with other surface antigens, perhaps on other
cells, is more likely to be the case with these receptors.
Because enhanced tyrosine phosphorylation has been shown to be responsible
for causing cellular transformation, underexpression, or inactivation, of
protein tyrosine phosphatases may also potentially result in oncogenesis.
For this reason, tyrosine-specific phosphatase genes are candidate
recessive oncogenes or tumor suppressor genes. In support of this theory,
the human RPTPase, RPTP.gamma., has been shown to map to a chromosomal
region, 3p14-21, which is frequently deleted in renal cell and lung
carcinomas (LaForgia, S. et al., 1991, Proc. Natl. Acad. Sci. USA
88:5036-5040). Recent studies, however, indicate that protein tyrosine
phosphatase action need not only be suppressive. It has been shown that
members of the src family of non-receptor tyrosine kinases contain
inhibitory tyrosine phosphorylation sites in the carboxy terminal tails
(reviewed by Hunter, T., 1987, Cell 49:1-14). When these sites are
phosphorylated, the molecules' tyrosine kinase activity is inhibited
(Nada, S. et al., 1991, Nature 351:69-72). It has further been
demonstrated that, in T cells, the dephosphorylation of such inhibitory
sites by a protein tyrosine phosphatase (CD45) leads to enhanced tyrosine
phosphorylation (Ledbetter, J. A. et al., 1989, Proc. Natl. Acad. Sci. USA
86:8628-8632), indicating, therefore, that phosphatases may function as
activating and well as inhibitory signaling enzymes. Also,
dephosphorylation of a tyrosine residue has been suggested to be an
obligatory step in the mitotic activation of the maturation-promoting
factor kinase (Morla, A. O. et al., 1989, Cell 58:193-203). Taken
together, the above observations suggest that PTPases may play an
important role in cellular control mechanisms, as effectors in mechanisms
of transmembrane signaling, as cell-cycle regulators, and as potential
oncogenes and anti-oncogenes.
3. SUMMARY OF THE INVENTION
The present invention relates to a new class of receptor protein tyrosine
phosphatase molecule, to the family of ligands that binds this new class
of receptor, and to the uses of such receptors and ligands. Specifically,
the members of this new class of receptor protein tyrosine phosphatase
molecule are proteoglycans and/or possess an extracellular carbonic
anhydrase structural domain. The characterization of one such receptor
molecule, RPTP.beta., is described in the working examples presented
herein.
The ligands which bind the receptor protein tyrosine phosphatases of the
invention may include, but are not limited to, tenascin and/or members of
the cell adhesion molecule (CAM) family of extracellular molecules. The
discovery that CAMs bind receptor protein tyrosine phosphatases represents
the first identification of a natural ligand for this type of receptor.
Binding of two CAMs, namely N-CAM and Ng-CAM, to the receptor protein
tyrosine phosphatases of the invention is demonstrated in the working
examples presented herein. In addition, as is demonstrated in the Working
Example presented below in Section 8, the receptor protein tyrosine
phosphatases of the invention also bind the extracellular matrix molecule
tenascin. The receptors and the receptor-binding ligands of the invention
may be used to develop compounds and strategies for modulating cellular
processes under the control of the receptor protein tyrosine phosphatases.
Such processes include, but are not limited to, normal cellular functions
such as differentiation, metabolism, cell cycle control, wound healing and
neuronal function; cellular behavior such as motility, migration, and
contact inhibition, in addition to abnormal or potentially deleterious
processes such as virus-receptor interactions, inflammation, cellular
transformation to a cancerous state, and the development of Type 2,
insulin Independent, diabetes mellitus. Compounds that may interfere with
ligand binding are described and methods for identifying other potential
ligands, such as CAM-type ligands, growth factors, or extracellular matrix
components, are discussed.
4. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. The amino acid sequence of RPTP.beta. [SEQ ID NO:2]. The protein
sequence of RPTP.beta. containing 2308 amino acids is indicated. The
hydrophobic signal peptide is underlined (amino acids 1-21), and the
transmembrane peptide is designated in bold-type (amino acids 1637-1662).
The 21 potential N-glycosylation sites are indicated by the arrows. The
CAH-related domain and
FIG. 2. Chromosomal localization of human RPTP.beta..
A. Presence of the RPTP.beta. gene in a panel of 17 rodent-human hybrids. A
completely stippled box indicates that the hybrid named in the left column
contains the chromosome indicated in the upper row; lower-right stippling
indicates presence of the long arm (or part of the long arm, indicated by
a smaller fraction of stippling) of the chromosome shown above the column;
upper left stippling indicates presence of the short arm (or partial short
arm) of the chromosome listed above the column; an open box indicates
absence of the chromosome above the column; the column for chromosome 7 is
boldly outlined and stippled to highlight correlation of presence of this
chromosome with the presence of the RPTP.beta. gene. The pattern of
retention of the RPTP.beta. sequences in the hybrids is shown to the right
where presence of the gene in the hybrids is indicated by a stippled box
with a plus sign and absence of the gene is indicated by an open box
enclosing a minus sign. B. RPTP.beta. maps to 7q31-q33. Chromosome in situ
hybridization of a 1.8 kb RPTP.beta. cDNA to normal human metaphases
confirmed localization of the gene to 7q and revealed a peak of grains
centered over region 7q31.3-7q32 as illustrated on the right to the
chromosome sketch. Each dot representing an autoradiographic grain.
FIG. 3. Analysis of the expression of RPTP.beta. in various murine tissues
and cell lines.
A. Poly A+ RNA (1 .mu.g per sample) from the various murine tissues
indicated were loaded onto a 1.0% agarose/2.2M formaldehyde gel and probed
with the per amplified murine DNA fragment, pBSMBDII (described in
Materials and Methods, Section 6.1.4). B. The blot in A. was stripped of
probe and rehybridized with a .sup.32 P labeled rat actin probe. C. 20
.mu.g of total cellular RNA (lanes 1-5) and 1 .mu.g of Poly A+ RNA (lane
6) isolated from the various glioblastoma and neuroblastoma cell lines
indicated were loaded onto on RNA gel and probed with a DNA fragment
isolated from the human brain stem cDNA clone that begins with sequences
just 5' of the transmembrane region and extends and includes all of the
sequences in phosphatase domain I.
FIG. 4. Northern blots to identify alternative splicing of RPTP.beta.
transcripts. A. A schematic diagram of the protein encoded by the full
length RPTP.beta. cDNA compared to the putative protein encoded by the two
independently isolated cDNA clones that carry an identical deletion of 258
bp in the extracellular region of the protein. The position of the
deletion is indicated by the dotted line with the number of the amino acid
that remains at both the 5' and 3' end of the deletion indicted. The
location of the two probes used in Northern analysis (probes 1 and 2) are
indicated. TM, transmembrane peptide; DI, phosphatase domain I and DII,
phosphatase domain II. B. Poly A+ RNA (1 .mu.g) isolated from the Lan 5
neuroblastoma cell line was separated on a RNA formaldehyde gel and probed
with human probe 1 (P1) that contains 1.3 kb of sequences derived from the
extreme 5' end of the cDNA clone and human probe 2 (P2) that contains 1.6
kb of sequences derived from the portion of the full length cDNA clone
that is deleted in clones BS-d14 and Cau-d11.
FIG. 5. In situ hybridization analysis of RPTP.beta. in developing and
adult mouse brain. A. A sagittal section through an embryonic day 20 (E20)
mouse shows that RPTP.beta. is preferentially expressed in the developing
central nervous system. The highest level of expression is seen in the
ventricular zone (VZ). B. A sagittal section through the adult mouse brain
shows discrete bands of expression in the Purkinje cell of the cerebellum,
the dentate gyrus (OG), and the anterior horn of the lateral ventricle
(AH).
FIG. 6. Identification of endogenous RPTP.beta. protein expression in Lan 5
cells. Immunoprecipitation of RPTP.beta. with normal rabbit serum (NRS,
lane 1) and immune RPTP.beta. antiserum (.alpha.PTP.beta., lanes 2 and 3)
from lysates of .sup.35 S methionine-labeled Lan 5 cells that had been
labeled in the absence (lanes 1 and 2) or presence of tunicamycin (lane
3). Apparent molecular weight is approximately 300 kD in the absence, and
250 kD is the presence, of tunicamycin. Immunoprecipitation of the EGF
receptor with RK2 antibody (.lambda.aEGFR, lanes 4 and 5) from lysates of
.sup.35 S methionine-labeled Lan 5 cells labeled in the absence (lane 4)
and presence (lane 5) of tunicamycin.
FIG. 7. Identification of a CAH-related domain in the extracellular region
of RPTP.beta.. A. The alignment of the amino acid sequence of the
CAH-related domain of RPTP.beta. with the corresponding domain of
RPTP.gamma. [SEQ ID NO:3] and the six different isoforms of CAH [SEQ ID
NOS:4-9] (Deutsch, H. F., 1987, Int. J. Biochem. 19:101-113). The amino
acid sequences that are boxed in black are those that are identical in all
six isoforms of CAH. The sequences that are boxed in the gray hatches are
those that are identical between the CAH-related domains of RPTP.beta. and
RPTP.gamma.. B. The percent similarity, taking into account conservative
substitutions of amino acids utilizing the program, between the
CAH-related domains of RPTP.beta. and RPTP.gamma. and the six isoforms of
CAH is indicted in this grid.
FIG. 8 Polyacrylamide gel of an immunoprecipitation, using .sup.35
S-NaSO.sub.4 -labeled cell lysates from 293 cells transfected with
RPTP.beta. cDNA (Lane 1) or from control, 293 cells transfected with
vector alone (Lane 2). Antiserum used was directed against RPTP.beta., as
described in Section 6.1.5.
FIG. 9 Polyacrylamide gel of an immunoprecipitation, using .sup.35
S-Met-labeled cell lysates from 293 cells transfected with RPTP.beta. cDNA
(Lane 1) or from control, 293 cells transfected with vector alone (Lane
2). Antiserum used was directed against RPTP.beta., as described in
Section 6.1.5.
FIG. 10 Polyacrylamide gel of an immunoprecipitation, using .sup.35
S-Met-labeled cell lysates from 293 cells transfected with RPTP.beta. DNA
(Lanes 3 and 4) or from control, 293 cells transfected with vector alone
(Lane 1 and 2). Lanes 2 and 4 represent lysates that have been
chondroitinase ABC-treated, while 1 and 3 are untreated lysates. Antiserum
used was directed against RPTP.beta., as described in Section 6.1.5.
FIG. 11. Effects of the proteoglycan 3F8 on aggregation of
Ng-CAM-Covaspheres. Green-fluorescing Ng-CAM-Covaspheres after incubation
for 2 hours at 25.degree. (A) in the presence of 10 .mu.g/ml of BSA. (B)
30 .mu.g/ml 3F8 proteoglycan. Covaspheres were visualized using a Nikon
Diaphot microscope equipped for fluorescence and were photographed using a
N2000 camera.
FIG. 12. Inhibition of NG-CAM-Covasphere aggregation by 3F8. The appearance
of superthreshold aggregates of Covaspheres coated with Ng-CAM was
measured after 2 hours using a Coulter counter. 6 .mu.l samples of
Ng-Covaspheres were mixed in a final volume of 60 .mu.l PBS in the
presence of various concentrations of native 3F8 proteoglycan (PG, solid
lines) and chondroitinase-treated 3F8 proteoglycan core (Chase, dashed
lines) from 7-day brain. Data represent means (N=3).+-. the standard error
of the % of the control levels of superthreshold aggregates detected. The
mean level of superthreshold particles in control samples was
32,582.+-.788.
FIG. 13. Inhibition of N-CAM-Covasphere aggregation by
chondroitinase-treated 3F8 (circles). The appearance of superthreshold
aggregates of Covaspheres coated with N-CAM was measured after 2 hours.
Chondroitinase-treated 3F8 used. Data represents a mean (N=3) of the %
control levels of superaggregates detected. The mean level of
superthreshold particles in control samples was 19,993+/-2,190.
FIG. 14. Comparison of the amino acid sequences of the carbonic anhydrase
domains contained in rat 3F8 [SEQ ID NO:10] and human RPTP.beta. proteins.
Top sequence represents the RPTP.beta. sequence, bottom line the 3F8
sequence.
FIG. 15. Binding of tenascin to 3F8 proteoglycan (PG). Panels (1-4) consist
of identical fields which were visualized specifically for
green-fluorescing Covaspheres (right) and red-fluorescing (left) tenascin
Covaspheres. Panel 1: tenascin Covaspheres mixed with green-fluorescing
3F8 PG Covaspheres; Panel 2: tenascin Covaspheres mixed with
green-fluorescing 3F8 PG Covaspheres in the presence of 3F8 monoclonal
antibodies; Panel 3: tenascin Covaspheres mixed with green-fluorescing
aggegrecan Covaspheres; Panel 4: tenascin Covaspheres mixed with green
fluorescing Ng-CAM Covaspheres.
5. DETAILED DESCRIPTION OF THE INVENTION
This invention involves a new class of receptor protein tyrosine
phosphatase molecule whose members are proteoglycans and/or possess an
extracellular carbonic anhydrase structural domain. In addition, two
classes of molecules, first, the extracellular matrix molecule tenascin,
and second, the cell adhesion family of molecules (CAMs), that bind to,
and act as ligands for, this new class of receptor are also described. The
discovery that CAMs bind receptor protein tyrosine phosphatases represents
the first identification of a natural ligand for this type of receptor.
Binding of two CAMs, namely N-CAM and Ng-CAM, to the receptor protein
tyrosine phosphatases of the invention is demonstrated in the working
examples presented herein. The further discovery that the extracellular
matrix molecule tenascin binds the receptor protein tyrosine phosphatases
of the invention identifies the second natural ligand for these receptors.
A number of uses for the receptors and the receptor-binding ligands of the
invention are also encompassed in the invention. Briefly, the receptor and
the receptor-binding ligands may be used to develop compounds and
strategies for modulating cellular processes under the control of the
receptor protein tyrosine phosphatases. Such processes include, but are
not limited to, normal cellular functions such as differentiation,
metabolism, cell cycle control, wound healing, and neuronal function;
cellular behaviors such as motility, migration, contact inhibition, and
signal transduction; in addition to abnormal or potentially deleterious
processes such as virus-receptor interactions, inflammation, cellular
transformation to a cancerous state, and the development of Type 2,
insulin independent diabetes mellitus. Compounds that may interfere with
ligand binding are described and methods for identifying other potential
ligands, such as CAM-type ligands, growth factors, or extracellular matrix
components, are discussed. Finally, working examples are presented in
which one member of the new RPTPase family of molecules, RPTP.beta., is
characterized, and additionally two members of the CAM family, N-CAM and
Ng-CAM, and the extracellular matrix molecule tenascin are all shown to
bind to the new RPTPase class of receptor.
5.1 RPTPases
The RPTPases of the invention that are proteoglycans may be modified with
macromolecules composed of glycosaminoglycan (GAG) chains (glycans)
covalently bound to the RPTPase protein core. GAG components may consist
of such units as hexosamine (D-glucosamine (GlcN) or D-galactosamine
(GalN)), and either hexuronic acid (HexA; D-glucuronic acid (GlcA) or
L-iduronic acid (IdoA)) or galactose units (as in keratin sulfate) that
are arranged in alternating, unbranched sequence, and carry sulfate
substituents in various positions. The glycan backbones of the RPTPase
molecules may include, but are not limited to, a basic structure composed
of (HexA-GalN).sub.n, (HexA-GlcN).sub.n, or (Gal-GlcN).sub.n disaccharide
units. While these structures connote the basic structure of the RPTPase
modifications, such modifications may also contain marked heterogeneity
within as well as between the individual polysaccharide chains. Such
heterogeneity is an expected byproduct of the mechanism of GAG
biosynthesis, and may include, but is not limited to differences in
sulfate substitutions along the chain and epimerization of one unit to
another (GlcA to IdoA, for example). At least one glycan chain must be
attached to the protein core of each proteoglycan RPTPase. Glycan chains
may, but are not required to, be attached to the protein core at the
serine (Ser) amino acid residue of the sequence, Ser-Gly-X-Gly [SEQ ID
NO:11], where Gly is a glycine amino acid residue and X is any amino acid
residue. Additionally, glycan chains may be attached to the protein core
at the serine residue in the sequence Z-Ser-Gly or Z-Gly-Ser, where Z is
an acidic amino acid residue.
The members of the RPTPase class of the invention may include an
extracellular stretch of amino acids that shares similarity with the known
carbonic anhydrase isoforms (Deutsch, H. F., 1987, Int. J. Biochem.
19:101-113). Such sequences need not have carbonic anhydrase enzymatic
activity. One or more complete or partial carbonic anhydrase motifs may be
present on a single RPTPase molecule. Within the CAH region of similarity
there may exist amino acid substitutions, as well as short amino acid
deletions, and/or short amino acid additions that diverge from the known
CAH isoforms. Such divergent sequences are acceptable as long as the
overall amino acid sequence similarity to CAH remains at least about 25%
and/or the tertiary structure or the domain remains similar to that of
CAH.
Presented in the working example in Section 6 is the characterization of
one member, RPTP.beta., of this new class of RPTPase molecule. In this
example, it is shown that RPTP.beta. not only contains a CAH-like domain
but is also a proteoglycan.
5.2 RPTPase LIGANDS
One class of molecules that acts as a preferred ligand for the receptors of
the invention is the cell adhesion family of molecules (CAMs). Such
molecules include, but are not limited to, any member of the classes of
Ca.sup.2+ -independent CAMs, cadherins, which are Ca.sup.2+ -dependent
CAMs, and integrins, which are Ca.sup.2+ - or Mg.sup.2+ -dependent CAMs.
Ca.sup.2+ -independent CAMs include such molecules as the N-CAM family,
Ng-CAM, L1, J1, Fasciclin III, and MAG molecules. The cadherins include
such molecules as N-cadherin, E-cadherin, P-cadherin, L-CAM, B-cadherin,
and T-cadherin. As is demonstrated in the working example presented in
Section 7, two members of the CAM family of molecules, N-CAM and Ng-CAM,
bind members of the RPTPase class of molecule described in this invention.
It had previously been speculated that receptor phosphatases themselves may
function as cell adhesion molecules because some of them contain motifs
such as IgG-like or fibronectin Type III repeats typical of CAMs. In
addition, because CAMs are known to undergo homotypic ("like" molecule)
binding, it had been proposed that PTPases with IgG and fibronectin motifs
may also under go homotypic interactions. It is of note, however, that
IgG-like and fibronectin motifs are found in many surface receptors and
proteins which do not undergo homotypic interactions. Contrary to this
proposal, though the working Example of Section 6 described herein
demonstrates that CAMs act as ligands for the RPTPase molecules of this
invention, which contain no IgG-like motifs. Thus, even in the absence of
peptide domain similarities, a ligand/receptor interaction does, in fact,
occur between the RPTPase class of molecule disclosed in this invention
and CAMs, where no interaction has previously been predicted to occur.
The ligands of the invention may be transmembrane proteins,
glycosylphosphatidylinositol-linked membrane proteins, or secreted
proteins. The molecules that constitute the ligands of this invention may
contain one or more peptide domains, including, but not limited to, one or
more Ig (immunoglobulin) domains (Williams, A. F., 1987, Immunol. Today
8:298-303), one or more fibronectin type III domains (Hynes, R. O., 1990,
Fibronectins, Springer-Verlag, N.Y.), and/or one or more ectodomains
(Takeichi, M., 1991, Science 251:1451-1455). Ig domains may share
characteristics with both immunoglobulin constant and variable regions.
Such characteristics may include pairs of cysteine residues, spaced
approximately 60 amino acids apart, that form disulfide bonds with each
other. Molecules may exhibit one or amino acid repeats of the sequence
DRE, DXNDN [SEQ ID NO:12], DXD, DVNE [SEQ ID NO:13], DXE, and/or DPD. If
the molecules are transmembrane proteins, such sequences should be present
in the extracellular portion of the molecule.
Because the RPTPase molecules of this invention may be proteoglycans,
several other non-CAM-like ligands may exist. For example, such
extracellular matrix molecules as vitronectin, fibronectin, and laminin
have been known to bind to the GAGs of certain proteoglycans. Also, growth
factors, such as fibro-blast growth factors, and Schwann cell growth
factor, have also been demonstrated to have affinity for proteoglycan GAG
chains. Therefore, molecules including, but not limited to extracellular
matrix molecules and growth factors are potential ligands for the RPTPase
class of molecule presented in this invention.
As is demonstrated in the Working Example presented below in Section 8, the
extracellular matrix molecule tenascin acts as a ligand for the RPTP.beta.
member of the RPTPase molecules of the invention. Therefore, the ligands
of the invention may further be all or any part of the tenascin
hexabrachion molecules that are capable of binding one or more of the
members of the RPTPase molecules of the invention. Such tenascin molecules
include molecules derived from human as well as other species, such as
mouse, chicken and the like, and further include tenascin molecules
comprising subunits encoded by any of the alternatively spliced tenascin
mRNAs. The portions of the tenascin molecules which may act as ligands of
the invention include, but are not limited to, any tenascin large subunit
and/or any tenascin small subunit. Further, the tenascin molecules which
act as ligands of the invention may include, but are not limited to, all
or any portion of the terminal knob domain, the thick distal segment,
and/or the thin proximal segment of any of the tenascin hexabrachion arms,
all or any portion of a tenascin T-junction domain corresponding to the
position wherein three tenascin hexabrachion arms join to form a trimer
and/or all or any portion of a tenascin central knob, corresponding to the
position wherein two tenascin trimers join to form a tenascin hexamer.
Other portions of tenascin molecules which may act as ligands of the
invention include all or any part of specific domains present as repeats
(i.e., greater than one copy per hexabrachion arm) within the tenascin
molecules. Such tenascin domain may include, but are not limited to,
EGF-like domains, tenascin domains similar to Type-III domains of
fibronectin, and/or tenascin carboxy-terminal domains similar to the
carboxy-terminal region of fibrinogen .beta. and .gamma. chains,
specifically that fibrinogen region which forms the globular domains of
the fibrinogen D-module (Erickson, H. P. and Fowler, W. E., 1983, Ann.
N.Y. Acad. Sci. 408: 146-163).
5.3 USES AND ADMINISTRATION OF RPTPase LIGANDS
Depending on the individual molecule, some RPTPase molecules may become
activated upon ligand binding, and others may become inactivated (the
activity referred to here being the RPTPases' phosphatase activity).
Ligand binding to RPTPase molecules may affect a variety of cellular
processes. Such processes include, but are not limited to, normal cellular
functions such as differentiation, metabolism, cell cycle control, wound
healing, and neuronal function; cellular behavior, such as cell motility,
migration, and contact inhibition; in addition to abnormal or potentially
deleterious processes such as virus-receptor interactions, inflammation,
cellular transformation to a cancerous state, and the development of Type
2, insulin independent diabetes mellitus. RPTPase/ligand binding may exert
an effect on the above-mentioned processes within the RPTPase-exhibiting
cell. In addition, because ligands of the invention, CAMs for example, are
often cell surface proteins, RPTPase/ligand binding may elicit an effect
on the ligand-exhibiting cell. Alternatively, RPTPases may contribute to
the control of such cellular processes by exerting an effect directly on
the ligand itself, via, for example, a ligand
phosphorylation/dephosphorylation reaction. The receptors and the
receptor-binding ligands of the invention may be used as drugs that can
modulate the cellular processes under the control of the RPTPases. In
addition, methods are presented below for the identification of compounds
that affect RPTPase activity, and such compounds may also be used as drugs
that can modulate one or more of the cellular processes mentioned above.
The receptors or their ligands may be used directly to modulate processes
such as those mentioned above. For example, soluble RPTPases may be
administered, using techniques well known to those skilled in the art,
that could act to compete with endogenous transmembrane receptor molecules
for available ligands, thus reducing or inhibiting ligand binding to
endogenous RPTPases. The effect of such a procedure would be to activate,
reduce or block the signal normally transduced into the cell (either the
RPTPase-exhibiting cell, or the ligand-exhibiting cell) via ligand binding
to transmembrane RPTPase. The RPTPases used here may include the entire
molecule or, alternatively, only the RPTPase extracellular domain, or a
part of the RPTPase extracellular domain thereof.
In addition, ligands may be administered, again, using techniques well
known to those in the art. Such administration would lead to a greater
than normal number of transmembrane RPTPases being bound by ligand,
potentially causing an amplification of the ligand-bound state within
cells exhibiting RPTPases. Alternatively, the administered ligand may be
composed of a modified form of said ligand such that receptor binding may
still occur, but the normal result of such binding (receptor activation or
inactivation, as the case may be) does not occur. A ligand with such a
design would act in much the same way that administration of soluble
RPTPase would, in that both procedures would have the final effect of
reducing the number of functionally bound RPTPase transmembrane molecules,
therefore lowering or blocking the normal extracellular signal being
transduced into the RPTPase-exhibiting cell via normal ligand binding to
transmembrane RPTPase. The effect on a ligand-exhibiting cell, for
example, one exhibiting cell surface CAMs, would also be the same in that
an overall lower number of endogenous ligands would be bound, therefore
lowering or blocking the effect of RPTPase binding on such
ligand-exhibiting cells.
Depending on the specific conditions being treated, agents may be
formulated and administered systemically or locally. Techniques for
formulation and administration may be found in "Remington's Pharmaceutical
Sciences," Mack Publishing Co., Easton, Pa., latest edition. Suitable
routes may include oral, rectal, transmucosal, or intestinal
administration; parenteral delivery, including intramuscular,
subcutaneous, intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or intraocular
injections, just to name a few. For injection, the agents of the invention
may be formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
RPTPases and/or their ligands may also be used to screen for additional
molecules that can act to modulate the activity of cellular processes such
as those described above. For example, compounds that bind to RPTPase
molecules may be identified. Such compounds may include, but are not
limited to anti-RPTPase antibodies. One method that may be pursued in the
isolation of such RPTPase-binding molecules would include the attachment
of RPTPase molecules to a solid matrix, such as agarose or plastic beads,
microtiter wells, or petri dishes, and the subsequent incubation of
attached RPTPase molecules in the presence of a potential RPTPase-binding
compound or compounds. After incubation, unbound compounds are washed
away, and the RPTP-bound compounds are recovered. In this procedure, large
numbers of types of molecules may be simultaneously screened for
RPTPase-binding activity. Bound molecules could be eluted from the RPTPase
molecules by, for example, competing them away from the RPTPase molecules
with the addition of excess ligand.
The effect of a compound on the phosphatase activity of RPTPase molecules
can also be determined. Such a compound may, for example, be one isolated
using a procedure such as the binding technique described above. One
method that may be utilized for determining the effects of a compound on
RPTPase phosphatase activity would involve exposing such a compound to a
preparation of cultured cells that express the RPTPase of the invention,
and subsequently measuring the phosphatase activity of the culture. The
compound of interest may be introduced to the cells, for example, by
addition of the compound to the tissue culture medium. The phosphatase
activity of the cells within the tissue culture preparation may be
determined by measuring the level of cellular phosphotyrosine within the
culture, using method that are well known in the art (Honegger et al.,
1987, Cell 51:199-209; Margolis et al., 1989, Cell 57:1101-1107). To
properly determine the effects of addition of the compound, the cellular
phosphotyrosine levels of the same type of tissue culture preparation that
has not been exposed to this compound must also be measured, and the two
levels must then be compared. For example, RPTPases may be incorporated
into apparatuses including but not limited to affinity columns such that
large numbers of molecules may be screened quickly by being applied to
said apparatuses. Those molecules with an affinity for RPTPases will be
bound. Such binding will also bring about a partial purification of the
molecules of interest. In order to continue the purification process, the
bound molecules should be eluted off the above described apparatuses, for
example by competing them away from the RPTPases with excess ligand, and
the process should be repeated until the molecule of interest is purified
to the extent necessary.
6. EXAMPLE: CHARACTERIZATION OF THE RECEPTOR PROTEIN TYROSINE PHOSPHATASE
RPTP.beta.
The subsections below describe the characterization of a human receptor
protein tyrosine phosphatase molecule, RPTP.beta.. It is shown that this
RPTP.beta. contains an extracellular carbonic anhydrase domain and is a
proteoglycan. In addition, it is shown that RPTP.beta. mRNA and protein
are predominantly expressed in brain tissue.
6.1 MATERIALS AND METHODS
6.1.1 ISOLATION OF cDNA CLONES AND DNA SEQUENCE ANALYSIS
A cDNA clone containing a portion of the coding sequences for RPTP.beta.
was isolated after screening a .lambda.gt11 human infant brain stem cDNA
library under conditions of reduced stringency with a nick translated LCA
probe that included both phosphatase domains (Kaplan, R. et al., 1990,
Proc. Natl. Acad. Sci. USA 87:7000-7004). Since the 5' end of this gene
was not present in the original clone, the library was rescreened with a
DNA fragment that was generated from the 5' end of the original clone. The
probe was labeled with .sup.32 P dCTP utilizing the random prime method
(USB) and hybridization was performed under moderately stringent
conditions at 42.degree. C. in a buffer containing 50% formamide,
5.times.SSC, 20 mM Tris-CL pH 7.6, 1.times. Denhardt's solution, 0.1% SDS
and 100 .mu.g/ml of sheared and denatured salmon sperm DNA. After
hybridization, phage filters were washed three times for 20 min at
50.degree. C. in a buffer containing 0.1.times.SSC/0.1% SDS and then were
processed for autoradiography. The brainstem library was rescreened a
total of three times in order to isolate overlapping cDNA clones that
contained the entire coding sequence for RPTP.beta..
CDNA inserts from positive recombinant plaque-purified were subcloned into
the plasmid vector, Blue Script (Stratagene), and sequenced by the dideoxy
chain termination method using the Sequenase Version 2.0 Kit (USB).
6.1.2 CHROMOSOMAL LOCALIZATION
Isolation, propagation and characterization of parental and somatic cell
hybrids used in this study have been described (Durst, M. et al., 1987,
Proc. Natl. Acad. Sci USA 84:1070-1074). Presence of specific human
chromosomes or regions of chromosomes has been confirmed by DNA
hybridization using probes for genes assigned to specific chromosome
regions. FIG. 2A depicts diagrammatically the chromosomes or partial
chromosomes retained in most of the hybrids used.
Chromosomal in situ hybridization was performed as described previously
(Cannizzano, L. A. et al., 1991, Cancer Res. 51:3818-3820). Slides
containing metaphase chromosomes from normal male (46 XY) peripheral blood
lymphocytes were aged at 4.degree. C. for 7-10 days and pretreated with
ribonuclease A (Sigma) for 1 hour at 37.degree. C. The chromosomal DNA was
denatured in a hybridization mixture containing 50% formamide, 2.times.SSC
and 10% dextran sulfate (pH 7.0). Hybridization was carried out at
37.degree. C. overnight. After rinsing at 39.degree. C. in three changes
of 50% formamide and 2.times.SSC, then five changes of 2.times.SSC, slides
were dehydrated, air dried, subjected to autoradiography, and banded with
Wright's-Giemsa stain solution mixed with 1-3 parts of pH 9.2 borate
buffer (Cannizzano, L. A. et al., 1991, Cancer Res. 51:3818-3820).
6.1.3 ISOLATION OF MOUSE SEQUENCES HOMOLOGOUS TO HUMAN RPTP.beta.
Two oligonucleotides in conserved phosphatase domain II were synthesized
according to the nucleotide sequence of human RPTP.beta.. These oligos, in
conjunction with phage DNA from a mouse brain cDNA library that was
purchased from Clonetech (Palo Alto, Calif.), were used in the polymerase
chain reaction with Taq polymerase (Perkin-Elmer) to amplify homologous
mouse RPTP.beta. sequences. The amplified product was purified and cloned
into the Blue Script plasmid vector (Stratagene, La Jolla, Calif.).
Homology was confirmed by DNA sequence analysis as described above. This
subcloned fragment will be referred to as PBSMBDII.
6.1.4 NORTHERN ANALYSIS
Total cellular RNA was prepared with the Strategene RNA isolation kit. Poly
A+ RNA was further selected utilizing oligo dT cellulose chromatography
(Stratagene). For Northern analysis, the RNA was separated on a 1.0%
agarose/2.2 M formaldehyde gel and transferred to a Nytran membrane
(Schleicher and Schuell) by capillary action. The membrane was
prehybridized and hybridized in 0.5 M sodium phosphate pH 7.2, 7% SDS, 1
mM EDTA, 100 .mu.g/ml salmon sperm DNA and then washed in 4 mM sodium
phosphate ph 7.2, 1% SDS, 1 mM EDTA at 65.degree. C. For the blot
containing RNA isolated from various mouse tissues, a .sup.32 P-labeled
probe was made utilizing pBSMBDII as template in the random prime labeling
reaction (USB). The human glioblastoma and neuroblastoma RNA blots were
probed with labeled restriction fragments isolated from different parts of
the human RPTP.beta. cDNA clones.
6.1.5 ANTIBODIES
A peptide derived from the carboxy-terminal 15 amino acids of human
RPTP.beta. was synthesized and coupled to Keyhole limpet hemocyanin
according to previously published procedures (Harlow, E. and Lane, D.,
1988, in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., pp. 77-88). This was used as immunogen to
inoculate two rabbits to produce polyclonal antisera against RPTP.beta..
Anti-EGF receptor immunoprecipitates were performed with RK2 antibody
which recognizes the glycosylated and nonglycosylated forms of the EGF
receptor (Kris, R. M. et al., 1985, Cell 40: 619-625).
6.1.6 CELL LABELING AND IMMUNOPRECIPITATION
The human neuroblastoma cell line, Lan 5, was maintained in Dulbecco's
modified Eagles medium (DMEM) contain 10% fetal bovine serum (FBS). Cell
treatment with tunicamycin involved incubating the cultures with 10
.mu.g/ml tunicamycin (Sigma) for 1 hour prior to .sup.35 S methionine
labeling. Treated and untreated cells were washed twice with methionine
free DMEM and then labeled for 4 hours with 0.15 mCi/ml .sup.32 S
methionine (purchased from New England Nuclear) in DMEM minus methionine
containing 1% dialyzed FBS. During the labeling period, 10 .mu.G/ml
tunicamycin was added to the medium of the treated cells. Cells were then
washed with ice cold phosphate-buffered saline and solubilized in a lysis
buffer containing Hepes (N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic
acid) pH 7.5, 150 mM NaCl, 1.0% Triton X-100, 10% glycerol, 1.5 mM
MgCl.sub.2, 1 mM ethylene glycol-bis (B-aminoethyl ether)-N, N, N',
N'-tetracetic acid (EGTA), 10 .mu.g leupeptin per ml, 1 mM
phenylmethylsulfunyl fluoride, and 10 .mu.g aprotinin per ml. For .sup.35
S-NaSO.sub.4 labeling, cells were washed twice with phosphate-buffered
saline (PBS), at which time labeling mix was added (NEX-041H medium plus
10% calf serum, gentamicin, and 200 .mu.Ci/ml of .sup.35 S-NaSO.sub.4).
Cells were labeled for 20 hours, washed twice with PBS, and solubilized as
described above. Cell lysates were clarified and then immunoprecipitated.
Lysates from .sup.35 S-Methionine-labeled cultures were immunoprecipitated
with normal rabbit antiserum, anti-RPTP.beta. antiserum or RK2 antiserum
for 2 hours at 4.degree. C. Lysates from .sup.35 S-NaSO.sub.4 -labeled
cultures were immunoprecipitated without preclearing, with anti-RPTP.beta.
antiserum for 2 hours at 4.degree. C. The immunocomplexes were then
precipitated with Protein A Sepharose (Sigma) for 45 min at 4.degree. C.
and washed 10 times with RIPA buffer (20 mM Tris-Cl ph 7.6, 300 mM NaCl, 2
mM EDTA, 1.0% Triton X-100, 1.0% sodium deoxycholate and 0.1% SDS). The
immunoprecipitated material was analyzed on SDS-polyacrylamide gels (7.5%
for .sup.35 S-Methionine, 5% for .sup.35 S-NaSO.sub.4) and then
fluorographed.
6.1.7 IN SITU HYBRIDIZATION ANALYSIS
Fresh frozen tissue was cut on a cryostat into 20 .mu.m thick sections and
thaw mounted onto gelatin coated slides. The sections were fixed in 4%
paraformaldehyde in 0.1 M sodium phosphate (pH 7.4) for 30 min and rinsed
3.times. for 5 min each in 0.1 M sodium phosphate and 1.times. for 10 min
in 2.times.SSC. The probe used in the hybridization analysis was a 49 base
oligonucleotide complementary to a portion of the RPTP.beta. mRNA in
phosphatase Domain II. The oligonucleotide was labeled with
[.alpha.-.sup.35 S] dATP (NEN Dupont) using terminal
deoxynucleotidyltransferase (Boerhinger Mannheim) and purified using
Sephadex G25 quick spin columns (Boerhinger Mannheim). The specific
activity of the labeled probes was between 5.times.10.sup.8
-1.times.10.sup.9 cpm/.mu.g. Prehybridizations and hybridizations were
carried out in a buffer containing 50% deionized formamide, 4.times.SSC,
1.times. Denhardt's, 500 .mu.g/ml denatured salmon sperm DNA, 250 .mu.g/ml
yeast tRNA and 10% dextran sulfate. The tissue was incubated for 12 h at
45.degree. C. in hybridization solution containing the labeled probe
(1.times.10.sup.6 cpm/section) and 10 mM dithiothreitol (DTT). Controls
for specificity were performed on adjacent sections by diluting the
labeled oligonucleotides with a 30 fold concentration of the appropriate
unlabeled oligonucleotide and by hybridization with a sense probe. After
hybridization the sections were washed in 2 changes of 2.times.SSC at room
temperature for 1 h, 1.times.SSC at 55.degree. C. for 30 min.,
0.5.times.SSC at 55.degree. C. for 30 min, 0.5.times.SSC at room
temperature for 15 min and dehydrated in 60%, 80%, and 100% ethanol. After
air drying, the sections were exposed to X-ray film for 5-10d.
6.2 RESULTS
6.2.1 THE PRIMARY AMINO ACID SEQUENCE OF RPTP.beta.
Four overlapping cDNA clones were isolated from the human brain stem
library that contained the entire coding sequence for RPTP.beta.. The
primary amino acid sequence deduced from the nucleotide sequence of the
cDNA clones is shown in FIG. 1. RPTP.beta. [SEQ ID NO:2] belongs to the
high molecular weight, transmembrane class of tyrosine phosphatases and is
encoded by 2308 amino acids. The protein contains a signal peptide
(underlined in FIG. 1) followed by a long extracellular domain of 1636
amino acids containing 21 potential N-glycosylation sites (indicated by
the arrows). A hydrophobic, transmembrane peptide (bold sequences) joins
the extracellular portion of the protein to two tandemly repeated and
conserved phosphatase domains (designated DI and DII).
6.2.2 CHROMOSOMAL LOCALIZATION OF HUMAN RPTP.beta.
The chromosomal localization of the human RPTP.beta. gene was initially
determined utilizing a panel of rodent-human hybrids carrying defined
human chromosomes or chromosome regions. DNAs from seventeen rodent-human
hybrids, carrying overlapping subsets of human chromosome regions
representing the entire human genome (see FIG. 2A) were cleaved with Eco
RI, electrophoresed, transferred to filters and hybridized to the
radiolabelled human RPTP.beta. probe. Results are summarized in FIG. 2A in
which it can be seen that the presence of human chromosome 7. A more
precise localization of the RPTP.beta. gene was determined by chromosomal
in situ hybridization to metaphase chromosomes of normal human
lymphocytes. This technique places the RPTP.beta. gene at 7q31-33 with the
most likely position at 7q31.3-7q32, which is diagrammatically shown to
the right of the chromosome 7 sketch in FIG. 2B.
6.2.3 NORTHERN BLOT ANALYSIS
Northern hybridization analysis of various murine tissue RNAs was performed
to determine the tissue specific expression of RPTP.beta.. The probe used
in this analysis was a portion of the murine homolog of RPTP.beta. that
was amplified in the polymerase chain reaction (see Materials and Methods)
and contains 405 nucleotides encoding 135 amino acids of Domain II. Based
on a nucleotide sequence comparison to the equivalent region of the human
cDNA clone, the murine and human clones are 88% identical at the
nucleotide level in this region of Domain II of RPTP.beta.. The results of
this Northern analysis (FIG. 3A) indicate the presence of two major
transcripts of 8.8 and 6.4 kb, respectively. A minor transcript of greater
than 9.4 kb is sometimes observed and might represent cross-reaction to a
highly related phosphatase. Both the transcripts are restricted to brain
tissue, and could not be detected in other tissue. The absence of
expression of RPTP.beta. in the majority of tissues is not due to degraded
RNA since the presence of intact actin transcripts was observed utilizing
the same blot (FIG. 3B).
Because the expression of RPTP.beta. was restricted to brain tissue,
expression of this phosphatase in different human glioblastoma cell lines
and a human neuroblastoma cell line, Lan 5, was examined. A human
RPTP.beta. probe hybridized to three major transcripts of 8.8, 7.5 and 6.4
kb respectively (FIG. 3C). These transcripts were only detected in RNA
isolated from the Lan 5 neuroblastoma cell line and were absent in the RNA
isolated from the four glioblastoma cell lines even though similar amounts
of total cellular RNA were loaded as determined from ethidium bromide
staining of the 28S and 18S ribosomal RNAs. The 8.8 and 6.4 kb transcripts
were identical in size to the two transcripts observed in RNA isolated
from mouse brain tissue (FIG. 3A). The presence of three transcripts in
Lan 5 RNA could be due to cross hybridization with other highly related
phosphatases since the probe used in this analysis was derived from
sequences in the conserved phosphatase Domain I or be due to alteratively
spliced RPTP.beta. transcripts. In order to obtain further insight into
the nature of the three RPTP.beta. transcripts, a similar Northern
analysis was performed on RNA isolated from Lan 5 cells with probes
isolated from the 5' portion of the human cDNA clones. The probes utilized
were derived from sequences in the extracellular domain that are unique
for RPTP.beta.. This analysis showed an identical pattern of hybridization
as what is observed in FIG. 3C. These results suggest that the three
distinct transcripts are products of alternatively spliced mRNAs.
6.2.4 IDENTIFICATION OF A VARIANT FORM OF RPTP.beta.
The overlapping human cDNA clones collectively contain greater than 8.1 kb
of coding and noncoding sequences and appear to represent the largest
transcript that is 8.8 kb in length. In screening the human brain stem
library and another human caudate library (Stratagene), two independent
cDNA clones, called Bsdl1 and Caudl1, were isolated that each contained an
identical deletion of 2581 nucleotides from the extracellular domain of
RPTP.beta.. This deletion did not introduce a stop codon or interrupt the
open reading frame of RPTP.beta. and joined amino acid 754 to amino acid
1615 as shown in FIG. 4A. The deleted clones maintained the same extreme
5' and 3' ends of the RPTP.beta. gene in addition to the sequences
encoding the transmembrane peptide and the two phosphatase domains. A
transcript corresponding to the deleted clone would be approximately 2.6
kb smaller than the transcript corresponding to the undeleted, full-length
clone. As shown in FIG. 3C, there is a transcript of 6.4 kb that is
approximately 2.4 kb smaller than the largest transcript which is 8.8 kb
in length. In order to determine whether the 6.4 kb transcript represents
the deleted form, Northern blot hybridization analysis was performed
utilizing RNA isolated from the Lan 5 cell line. Duplicate blots were made
from this RNA and hybridized with two distinct probes. One probe (probe 1)
was derived from sequences in the 5' end of RPTP.beta. that are present in
the deleted and full length cDNA clones. The other probe (probe 2)
encompasses the sequences that are no longer present in the deleted cDNA
clones. The location of probes A and B in the full length RPTP.beta. cDNA
is shown in FIG. 4A. Comparison of the Northern analysis with the two
probes revealed that probe 1 hybridized to the three distinct transcript
(FIG. 4B, P1) whereas probe 2 hybridized to the 7.5 and 8.8 kb transcripts
but failed to hybridize to the 6.4 kb transcript (FIG. 4B, P2). These
results provide preliminary evidence that the 6.4 kb transcript represents
a deleted and alternatively spliced form of RPTP.beta.. The nature of the
7.5 kb transcript remains to be determined.
6.2.5 IN SITU HYBRIDIZATION ANALYSIS
In order to obtain further insight into the expression of RPTP.beta., an in
situ hybridization analysis was performed to look for the expression of
RPTP.beta. in the developing mouse embryo. Studies were also performed to
determine whether RPTP.beta. gene expression is diffuse or restricted to
specific regions of the adult brain. The results of this analysis confirm
that RPTP.beta. is preferentially expressed in the central nervous system.
In the day 20 mouse embryo (E20), high level of expression was observed in
the ventricular zone of the brain (FIG. 5A.) and in the spinal cord. A
similar pattern of expression, with variable levels of intensity, has been
seen from embryonic day 13 to postnatal day 7. The level of expression is
much lower in the adult brain, and is discretely localized to the Purkinje
cell layer of the cerebellum, the dentate gyrus, and the anterior horn of
lateral ventricle (FIG. SB). The addition of a 30 fold excess of unlabeled
oligonucleotide completely blocked the labeling in all of these areas
indicating that this probe is hybridizing to mRNA in a sequence specific
manner. Results from the Northern blot and in situ hybridization analyses
demonstrate that RPTP.beta. has a restricted tissue specificity to
specific regions in the central nervous system and therefore may play an
important role in the development of the nervous system.
6.2.6 ENDOGENOUS RPTP.beta. PROTEIN EXPRESSION
Since RPTP.beta. transcripts were identified in the Lan 5 neuroblastoma
cell line, these cells were subsequently used to detect endogenous protein
expression. Cell lysates prepared from cultures labeled with .sup.35
S-methionine for 4 hours were immunoprecipitated with normal rabbit serum
or anti-RPTP.beta. antiserum (FIG. 6). A protein with apparent weight of
approximately 300 kd was recognized by the immune but not by the normal
rabbit serum (lanes 1 and 2). Since there are 21 potential N-glycosylation
sites, it was necessary to determine whether the protein
immunoprecipitated by the anti-RPTP.beta. antiserum represented the core
protein or a glycosylated form of the protein. In order to do this,
tunicamycin was added to the cells during the .sup.35 S-methionine
labeling period. The effects of tunicamycin treatment on RPTP.beta.
mobility was compared to the cell line were drug's ability to inhibit the
glycosylation of the EGF receptor, which is also expressed in this cell
line. Untreated cell lysates and lysates prepared from cells treated with
tunicamycin were immunoprecipitated with an antibody (RK2) that recognizes
the 170 kd glycosylated form and the 135 kd nonglycosylated form of the
EGF receptor. (Kris, R. M. et al., 1985, Cell 40:619-625; and FIG. 6,
lanes 4 and 5). The protein immunoprecipitated with anti RPTP.beta.
antiserum from Lan 5 cells, that had been metabolically labeled in the
presence of tunicamycin, migrated faster than the immunoprecipitated
product isolated from the untreated cells (compare lanes 3 and 2). The
molecular weight of the protein detected in FIG. 6, lane 3, is
approximately 250 kD a value consistent with that of the core protein
whose predicted molecular weight as deduced from the amino acid sequence
is approximately 254 kd.
6.2.7 THE PRESENCE OF CARBONIC ANHYDRASE RELATED SEQUENCES IN THE
EXTRACELLULAR DOMAIN OF RPTP.beta.
A stretch of 283 amino acids in the extracellular domain of a related
transmembrane-type phosphatase, RPTP.gamma. that shares homology with
different isoforms of the enzyme carbonic anhydrase (CAH) has recently
been identified. A similar stretch of CAH-related amino acids in
RPTP.beta. has now been identified at the extreme amino terminus of the
protein (designated as CAH-like in FIG. 1). Alignment of the CAH-related
domains of RPTP.beta. [SEQ ID NO:2] and RPTP.gamma. [SEQ ID NO:3] with the
six known isoforms of CAH [SEQ ID NOS:4-9] is shown in FIG. 7A. FIG. 7B
shows the percent similarity, taking into account conservative amino acid
substitutions between the CAH-related domain of RPTP.beta. and the
corresponding domain of RPTP.gamma. and the six CAH enzymes. The
CAH-related domain of RPTP.beta. ranges anywhere from 45-50% in amino acid
sequence similarity to the six different isoforms of CAH. The highest
degree of similarity (58%) exists between the CAH-related sequences of
RPTP.beta. and RPTP.gamma.. Therefore it appears that RPTPases, .beta. and
.gamma., may represent a new subgroup of tyrosine phosphatases that will
be characterized by the presence of CAH-related sequences in their
extracellular domains.
6.2.8 RPTP.beta. IS A PROTEOGLYCAN
In this series of experiments, it is demonstrated that RPTP.beta. exhibits
the characteristics of a proteoglycan. Specifically, it is shown that the
RPTP.beta. protein is covalently modified with high molecular weight,
sulfate-containing moieties, and that such moieties are sensitive to
chondroitinase ABC treatment.
6.2.8.1 .sup.35 SULFATE LABELING
In order to begin to address what post-translational modifications
RPTP.beta. undergoes, 293 cells transfected with RPTP.beta. DNA as well as
control, 293 cells transfected with vector alone were .sup.35 S-NaSO.sub.4
labeled. Immunoprecipitations of the lysates, using an anti-RPTP.beta.
antiserum were then performed. The gel illustrated in FIG. 8 shows the
results of one such immunoprecipitation. As can be seen in lane 1, a
significant amount of labeled material that reacts with the RPTP.beta.
antiserum does not enter the running portion of the gel. This is easily
contrasted to the non-transfected control lysate in lane 2, in which no
such material is detectable.
6.2.8.2 .sup.35 S-METHIONINE LABELING
In continuing to investigate the posttranslational modifications that
RPTP.beta. undergoes, 293 cells transfected with RPTP.beta. DNA as well as
control 293 cells transfected with vector alone were .sup.35 S-methionine
labeled. Immunoprecipitations of the lysates, using an anti-RPTP.beta.
antiserum were then performed. The gel illustrated in FIG. 9 shows the
results of one such immunoprecipitation. Lane 1 contains a large amount of
labeled material that reacted with the anti-RPTP.beta. antiserum which
does not enter the running portion of the gel and a significant amount
that does not even enter the stacking portion of the gel. By contrast,
lane 2, which contains the control lysate, exhibits no such material.
6.2.8.3 CHONDROITINASE TREATMENT
293 cells transfected with RPTP.beta. DNA as well as control 293 cells
transfected with vector alone were .sup.35 S-methionine labeled. Lysates
were immunoprecipitated using an anti-RPTP.beta. antiserum and then
chondroitinase ABC treated for 1 hour. The gel illustrated in FIG. 10
shows the results of one such immunoprecipitation. Lane 3 and 4 contain
non-treated and treated RPTP.beta.-transfected lysates, respectively. As
can be seen, the bulk of the labeled material that had not entered the gel
in the non-treated sample is absent in the treated sample, and in its
place, a labeled band of a lower molecular weight has appeared. In lanes 1
and 2 are non-treated and treated control lysates, respectively. No such
shift in high molecular weight labeled material is detectable here.
7. EXAMPLE: THE CELL ADHESION MOLECULES, N-CAM AND Ng-CAM ARE LIGANDS OF
THE RECEPTOR PROTEIN TYROSINE PHOSPHATASE, RPTP.beta.
The experiments described below demonstrate that a receptor protein
tyrosine phosphatase, the human RPTP.beta. molecule, binds the cell
adhesion molecules N-CAM and Ng-CAM. Section 7.2.1 demonstrate that the
rat proteoglycan, 3F8, binds these two CAM molecules. Section 7.2.2,
demonstrates that the carbonic anhydrase domains of rat 3F8 and human
RPTP.beta. are nearly identical, leading to the conclusion that RPTP.beta.
is the human counterpart of the rat proteoglycan 3F8. When taken together,
these two pieces of information, indicate that RPTP.beta. also binds the
two CAM molecules, N-CAM and Ng-CAM.
7.1 MATERIALS AND METHODS
7.1.1 PROTEINS AND ANTIBODIES
Ng-CAM and N-CAM were purified from 14-d embryonic chicken brains by
immunoaffinity chromatography using specific monoclonal antibodies
(Grumet, M. and Edelman, G. M., 1988, J. Cell Biol. 106:487-503). Analysis
of the proteins on SDS/PAGE showed that Ng-CAM consisted of a major
component of 135 kDa and lesser amounts of the 200 kDa and 80 kDa species
as described (Grumet, M. and Edelman, G. M., 1988, J. Cell Biol.
106:487-503) and N-CAM ran as hetero-disperse species above 12 kDa as
described previously (Hoffman, S. et al., 1982, J. Biol. Chem.
257:7720-7729).
Chondroitin sulfate proteoglycans were extracted with PBS from the brains
of 7-day or 2- to 2-month old Sprague-Dawley rats, and purified by ion
exchange chromatography and gel filtration (Kiang, W.-L. et al., 1981, J.
Biol. Chem. 256:10529-10537). 3F8 proteoglycan was then isolated by
immunoaffinity chromatography, using monoclonal antibodies coupled to
CNBr-activated Sepharose 4B (Rauch, U. et al., 1991, J. Biol. Chem.
266:14785-14801). Analysis of the proteins on SDS-PAGE following
chondroitinase-treatment showed that the core glycoprotein obtained by
chondroitinase treatment of the 3F8 proteoglycan from either early
postnatal or adult brain migrated on SDS-PAGE as a single bad at 400 kDa
(Rauch, U. et al., 1991, J. Biol. Chem. 266:14785-14801).
For studies of the core proteins, proteoglycans were digested for 45-60 min
at 37.degree. C. with protease-free chondroitinase ABC (Seikagaku America
Inc., Rockville, Md.) in 100 mM Tris-HCl buffer (pH 8.0 at 37.degree. C.)
containing 30 mM sodium acetate. A ratio of 1.5 mM chondroitinase/.mu.g
proteoglycan protein was used for the 3F8 proteoglycan. Completeness of
digestion was confirmed by SDS-PAGE, which demonstrated that the large
native proteoglycan which did not enter the separating gel was converted
to discrete core glycoprotein bands after enzyme treatment (Rauch, U. et
al., 1991, J. Biol. Chem. 266:14785-14801).
Polyclonal rabbit antibodies raised against chicken Ng-CAM were prepared as
previously described (Grumet, M. et al., 1984, Proc. Natl. Acad. Sci USA
81:267-271).
7.1.2 COVASPHERE AGGREGATION
Proteins (50 .mu.g) were covalently coupled to 200 .mu.l of 0.5-.mu.m
Covaspheres (Duke Scientific Corp., Palo Alto, Calif.), washed twice in
PBS containing 1 mg/ml BSA/10 mM NaN.sub.3, and resuspended in 200 .mu.l
of buffer as previously described (Grumet, M. and Edelman, G. M., 1988, J.
Cell Biol. 106:487-503). Quantitative measurements indicated that under
these conditions approximately 20% of the Ng-CAM protein was bound to the
Covaspheres. For Covasphere aggregation experiments, prior aggregates in
the bead preparations were first dissociated by sonication for 10-20 sec,
and 6 .mu.l aliquots (containing about 0.3 .mu.g of protein) were mixed
with 54 .mu.g of PBS containing the indicated amounts of proteins. After a
30 min incubation on ice, the samples were resonicated and aggregation was
monitored at 25.degree. C. The appearance of superthreshold aggregates of
Covaspheres was measured using a Coulter Counter (Model ZBI) fitted with a
100 .mu.m aperture set at amplication=0.17, aperture current=0.33,
threshold 10-100; these settings allowed detection of particles >.sup.- 4
.mu.m as described previously (Grumet, M. and Edelman, G. M., 1988, J.
Cell Biol. 106:487-503). Superthreshold particles were measured in samples
of 20 .mu.l that were diluted into 20 ml of PBS. To test the sensitivity
of proteoglycans to proteolysis, solutions containing 0.1 mg/ml
proteoglycan were treated with 10 .mu.g/ml of trypsin for 1 h at
37.degree. C. and the reaction was terminated by addition of 20 .mu.g/ml
of soybean trypsin inhibitor.
7.1.3 DNA SEQUENCING
Sequencing was performed according to standard dideoxy techniques.
7.2 RESULTS
7.2.1 THE RAT PROTEOGLYCAN, 3F8, BINDS CAMS
In previous studies, it was found that Ng-CAM (Grumet, M. and Edelman, G.
M., 1988, J. Cell Biol. 106:487-503) and N-CAM (Hoffman, S. and Edelman,
G. M., 1983, Proc. Natl. Acad. Sci. USA 80:5762-5766) individually
mediated homophilic binding when reconstituted into liposomes or when
covalently bound to the surface of beads (Covaspheres). The rate of
aggregation of Ng-CAM-Covaspheres was measured using a Coulter Counter to
detect aggregates larger than a given size and was shown to be highly
dependent on the concentration of Covaspheres in suspension. It was
previously determined that at a concentration of .about.85 cm.sup.2 of
bead surface area/ml, the appearance of aggregates began to reach a
plateau level after .about.1 h of incubation (Grumet, M. and Edelman, G.
M., 1988, J. Cell Biol. 106:487-503). Therefore, to explore potential
interaction between proteoglycans and CAMs, the effects of various
proteins and proteoglycans on the rate of aggregation of
Ng-CAM-Covaspheres were tested. Whereas control proteins including BSA and
fibronectin did not inhibit aggregation of the Ng-CAM-coated beads, one
distinct chondroitin sulfate proteoglycan, 3F8, inhibited aggregation
(FIG. 11). In contrast, aggrecan, a rat chondrosarcoma chondroitin sulfate
proteoglycan (Doege, K. M. et al, 1987, J. Biol. Chem. 262:17757-17767)
did not inhibit the aggregation, indicating that the effects were not
simply related to the presence of chondroitin sulfate. This conclusion was
further supported by the finding that the core glycoproteins obtained by
chondroitinase treatment of the proteoglycans were equally effective in
inhibiting the aggregation of Ng-CAM-Covaspheres (FIG. 12). In contrast,
most of the inhibitory activity was eliminated after treating the
proteoglycans with trypsin (see Materials and Methods, Section 7.1.2).
These results demonstrate that the effects of the 3F8 proteoglycan on
Ng-CAM binding are not mediated by the glycosaminoglycan chains, but that
a protein domain (or possibly a cluster of oligosaccharides present on the
proteoglycan core protein) is involved. Based on these results, all
further experiments were performed using chondroitinase-treated
proteoglycans.
The 3F8 proteoglycan inhibited aggregation of Ng-CAM-Covaspheres at 30
.mu.g/ml (FIG. 11). It is unlikely that the proteoglycans inhibited
Covasphere aggregation by a trivial mechanism such as proteolysis of
Ng-CAM because it was found that incubation of the 3F8 proteoglycan with
Ng-CAM for 1 h at 37.degree. C. had no effect of the molecular sizes of
the Ng-CAM components when resolved by SDS-PAGE.
To compare the effects of different proteoglycans we measured the
appearance of superthreshold aggregates of Covaspheres using a Coulter
Counter to detect aggregates larger than a given size. The aggregation of
Ng-CAM-Covaspheres was inhibited in a concentration-dependent manner by
the 3F8 proteoglycan (FIG. 12).
To determine whether the proteoglycans could affect other CAMs, a similar
series of experiments was performed using N-CAM coated beads. The
aggregation of N-CAM-Covaspheres was inhibited in a
concentration-dependent manner in the presence of the 3F8 proteoglycan
(FIG. 13). These results are similar to those obtained using
Ng-CAM-Covaspheres, (compare to FIGS. 11 and 12).
The inhibitory effect of 3F8 proteoglycan on the aggregation of Ng-CAM- and
N-CAM-coated beads were maximal at approximately 10 .mu.g/ml. In a typical
assay (60 .mu.l) at this concentration of proteoglycans, the amount of
proteoglycan in solution was 0.6 .mu.g and the amount of Ng-CAM on the
Covaspheres was approximately 0.3 .mu.g (see Materials and Methods,
Section 7.1.2), suggesting that the brain proteoglycan can perturb
homophilic Ng-CAM binding at approximately stoichiometric levels with
Ng-CAM.
7.2.2 RPTP.beta. REPRESENTS THE HUMAN COUNTERPART OF THE RAT PROTEOGLYCAN,
3F8
Comparison of the sequence of human RPTP.beta. with a partial sequence of a
proteoglycan designated 3F8 [SEQ ID NO:10] cloned from a rat brain stem
library (R. Margolis, personal communication), reveals that these two
proteins contain carbonic anhydrase-like domains and are 91.9% identical
at the amino acid level (FIG. 14). The maximum amino acid sequence
identity between the different members of the carbonic anhydrase family of
enzymes is 64%. This sequence information indicates that the two proteins,
the murine proteoglycan 3F8 and the human proteoglycan RPTP.beta., are
counterparts of each other.
8. EXAMPLE: THE EXTRACELLULAR MATRIX MOLECULE TENASCIN IS A LIGAND OF THE
RECEPTOR PROTEIN TYROSINE PHOSPHATASE RPTP.beta.
The experiments described below demonstrate that a receptor protein
tyrosine phosphatase, the human RPTP.beta. molecule, binds the
extracellular matrix molecule tenascin. Section 8.2 demonstrates that the
rat proteoglycan, 3F8, binds tenascin. Section 7.2.2, above, demonstrates
that the carbonic anhydrase-like domains of rat 3F8[SEQ ID NO:10] and of
human RPTP.beta.[SEQ ID NO:2] are nearly identical, leading to the
conclusion that RPTP.beta. is the human counterpart of the rat
proteoglycan 3F8. When taken together, these two pieces of information
indicate that RPTP.beta. also binds tenascin molecules.
8.1 MATERIALS AND METHODS
8.1.1 PROTEINS AND ANTIBODIES
Tenascin (Telios) purity was determined by SDS-PAGE gel analysis.
3F8 chondroitin sulfate proteoglycan. Rat brain 3F8 chondroitin sulfate
proteoglycan was prepared according to Rauch et al. (Rauch, U. et al.,
1991, J. Biol. Chem. 266:14785-14801).
Ng-CAM . Chicken brain Ng-CAM was purified as described in Grumet and
Edelman (Grumet, M. and Edelman, G.M., 1988, J. Cell Biology 106:487-503).
Briefly, Ng-CAM was purified from detergent extracts of 14 day embryo
brains by immunoaffinity chromatography using monoclonal antibodies (3G2;
Rieger, F. J. et al., 1986, J. Cell Biol. 103:379-391) against Ng-CAM. 1
mM PMSF (phenylmethylsulfonyl fluoride) was added to retard proteolysis.
Aggrecan. Aggrecan, a chondroitin sulfate proteoglycan derived from
cartilage, was prepared according to Grumet et al. (Grumet, M. et al.,
1993, J. Cell Biol. 120:815-824). Briefly, the chondroitin sulfate PG was
extracted from a transplantable rat chondrosarcoma (Choi, H. U. et al.,
1971, Proc. Natl. Acad. Sci. USA 68:877-879) and isolated by CsCl density
gradient centrifugation (Faltz, L. L. et al., 1979, J. Biol. Chem.
254:1375-1380).
3F8 monoclonal antibody. Prepared according to Rauch et al., 1991, J. Biol.
Chem. 266:14785-14801.
8.1.2 COVASPHERE PREPARATION AND AGGREGATION
Covaspheres were prepared as described in Grumet et al., 1993, J. Cell
Biol. 120:815-824. Briefly, proteins (50 .mu.g) were covalently coupled to
200 .mu.l 0.5 .mu.m Covaspheres (Duke Scientific Corp., Palo Alto,
Calif.), washed twice in PBS containing 1 mg/ml BSA, and 10 mM NaN.sub.3,
and resuspended in the original buffer volume. Covaspheres as supplied by
the manufacturer were at a concentration of 850-cm.sup.2 surface area/ml.
For Covasphere aggregation experiments, pre-existing aggregates in the bead
preparations were first dissociated by sonication for 10-20 seconds. After
a 30 minute incubation on ice, samples were resonicated and aggregation
was monitored at 25.degree. C.
Fluorescent Covaspheres were visualized using a Nikon Diaphot with a filter
capable of discriminating between fluorescein and rhodamine.
8.2 RESULTS
In the experiments presented in this Section, representative results of
which are presented in FIG. 15, it is shown that the proteoglycan 3F8
binds the extracellular matrix molecule tenascin. For these experiments,
red-fluorescing tenascin-coated Covaspheres were prepared as described in
Section 8.1.1, above, and mixed with various green-fluorescing
Covaspheres. Such green-fluorescing Covaspheres were coated with either
3F8 chondroitin sulfate brain proteoglycan (PG), aggrecan (a chondroitin
sulfate PG derived from cartilage), or Ng-CAM (a neural cell adhesion
molecule). All green-fluorescing Covaspheres were prepared according to
the methods described, above, in Section 8.1.1.
First, tenascin-coated Covaspheres were mixed with 3F8 PG-coated
Covaspheres, either in the absence (Panel 1) or presence (Panel 2) of a
3F8 monoclonal antibody. As can be seen in FIG. 15, Panel 1,
tenascin-coated Covaspheres (left, red-fluorescing) and 3F8 PG-coated
Covaspheres (right, green-fluorescing) aggregates are nearly identical,
indicating that tenascin binds 3F8 PG. FIG. 15, Panel 2 illustrates that
the tenascin-coated Covasphere/3F8 PG-coated Covasphere binding and
coaggregation is disrupted in the presence of 3F8 PG monoclonal antibody.
The use of a nonimmune antibody had no effect on tenascin/3F8 Covasphere
coaggregation, indicating the specificity of tenascin/3F8 PG interaction.
Tenascin-coated Covaspheres were also mixed with aggrecan-coated
Covaspheres the results of which are shown in FIG. 15, Panel 3. As can be
seen, little or no self-aggregation was observed for the red-fluorescing
tenascin-coated Covaspheres (left) or the green-fluorescing
aggrecan-coated Covaspheres (right). In addition, no co-aggregation was
detected between the red and green fluorescing Covaspheres. Thus, tenascin
binding to 3F8 PG is not due to merely an indiscriminate affinity for
proteoglycan molecules, but is specific for, at a minimum, this class of
RPTPase molecules. The specificity of the 3F8/tenascin interaction is
further demonstrated when tenascin-coated Covaspheres are mixed with
Ng-CAM-coated Covaspheres. As shown in FIG. 15, Panel 4, red-fluorescing
tenascin-coated Covaspheres (left) segregate independent of
green-fluorescing Ng-CAM fluorescing Covaspheres, indicating that tenascin
does not bind Ng-CAM, another molecule which has been shown to be a ligand
for 3F8 (see Section 7, above). Note that, as has been noted previously
(Grumet, M. and Edelman, G. M., 1988, J. Cell Biol. 106: 487-503), Ng-CAM
has the ability to self-aggregate, explaining the Ng-CAM-gated Covaspheres
aggregation in the present experiment (FIG. 15, Panel 4, right).
It is apparent that many modifications and variations of this invention as
set forth here may be made without departing from the spirit and scope
thereof. The specific embodiments described hereinabove are given by way
of example only and the invention is limited only by the terms of the
appended claims.
__________________________________________________________________________
# SEQUENCE LISTING
- - - - (1) GENERAL INFORMATION:
- - (iii) NUMBER OF SEQUENCES: 13
- - - - (2) INFORMATION FOR SEQ ID NO:1:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: peptide
- - (ix) FEATURE:
(A) NAME/KEY: Modified-sit - #e
(B) LOCATION: 1
(D) OTHER INFORMATION: - #/label= Xaa
/note= - #"Xaa = Ile or Val"
- - (ix) FEATURE:
(A) NAME/KEY: Modified-sit - #e
(B) LOCATION: 4
(D) OTHER INFORMATION: - #/label= Xaa
/note= - #"Xaa = Any amino acid"
- - (ix) FEATURE:
(A) NAME/KEY: Modified-sit - #e
(B) LOCATION: 7..8
(D) OTHER INFORMATION: - #/label= Xaa
/note= - #"Xaa = Any amino acid"
- - (ix) FEATURE:
(A) NAME/KEY: Modified-sit - #e
(B) LOCATION: 10
(D) OTHER INFORMATION: - #/label= Xaa
/note= - #"Xaa = Ser or Thr"
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
- - Xaa His Cys Xaa Ala Gly Xaa Xaa Arg Xaa Gl - #y
1 5 - # 10
- - - - (2) INFORMATION FOR SEQ ID NO:2:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2308 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
- - Met Arg Ile Leu Lys Arg Phe Leu Ala Cys Il - #e Gln Leu Leu Cys Val
1 5 - # 10 - # 15
- - Cys Arg Leu Asp Trp Ala Asn Gly Tyr Tyr Ar - #g Gln Gln Arg Lys Leu
20 - # 25 - # 30
- - Val Glu Glu Ile Gly Trp Ser Tyr Thr Gly Al - #a Leu Asn Gln Lys Asn
35 - # 40 - # 45
- - Trp Gly Lys Lys Tyr Pro Thr Cys Asn Ser Pr - #o Lys Gln Ser Pro Ile
50 - # 55 - # 60
- - Asn Ile Asp Glu Asp Leu Thr Gln Val Asn Va - #l Asn Leu Lys Lys Leu
65 - #70 - #75 - #80
- - Lys Phe Gln Gly Trp Asp Lys Thr Ser Leu Gl - #u Asn Thr Phe Ile His
85 - # 90 - # 95
- - Asn Thr Gly Lys Thr Val Glu Ile Asn Leu Th - #r Asn Asp Tyr Arg Val
100 - # 105 - # 110
- - Ser Gly Gly Val Ser Glu Met Val Phe Lys Al - #a Ser Lys Ile Thr Phe
115 - # 120 - # 125
- - His Trp Gly Lys Cys Asn Met Ser Ser Asp Gl - #y Ser Glu His Ser Leu
130 - # 135 - # 140
- - Glu Gly Gln Lys Phe Pro Leu Glu Met Gln Il - #e Tyr Cys Phe Asp Ala
145 1 - #50 1 - #55 1 -
#60
- - Asp Arg Phe Ser Ser Phe Glu Glu Ala Val Ly - #s Gly Lys Gly Lys
Leu
165 - # 170 - # 175
- - Arg Ala Leu Ser Ile Leu Phe Glu Val Gly Th - #r Glu Glu Asn Leu Asp
180 - # 185 - # 190
- - Phe Lys Ala Ile Ile Asp Gly Val Glu Ser Va - #l Ser Arg Phe Gly Lys
195 - # 200 - # 205
- - Gln Ala Ala Leu Asp Pro Phe Ile Leu Leu As - #n Leu Leu Pro Asn Ser
210 - # 215 - # 220
- - Thr Asp Lys Tyr Tyr Ile Tyr Asn Gly Ser Le - #u Thr Ser Pro Pro Cys
225 2 - #30 2 - #35 2 -
#40
- - Thr Asp Thr Val Asp Trp Ile Val Phe Lys As - #p Thr Val Ser Ile
Ser
245 - # 250 - # 255
- - Glu Ser Gln Leu Ala Val Phe Cys Glu Val Le - #u Thr Met Gln Gln Ser
260 - # 265 - # 270
- - Gly Tyr Val Met Leu Met Asp Tyr Leu Gln As - #n Asn Phe Arg Glu Gln
275 - # 280 - # 285
- - Gln Tyr Lys Phe Ser Arg Gln Val Phe Ser Se - #r Tyr Thr Gly Lys Glu
290 - # 295 - # 300
- - Glu Ile His Glu Ala Val Cys Ser Ser Glu Pr - #o Glu Asn Val Gln Ala
305 3 - #10 3 - #15 3 -
#20
- - Asp Pro Glu Asn Tyr Thr Ser Leu Leu Val Th - #r Trp Glu Arg Pro
Arg
325 - # 330 - # 335
- - Val Val Tyr Asp Thr Met Ile Glu Lys Phe Al - #a Val Leu Tyr Gln Gln
340 - # 345 - # 350
- - Leu Asp Gly Glu Asp Gln Thr Lys His Glu Ph - #e Leu Thr Asp Gly Tyr
355 - # 360 - # 365
- - Gln Asp Leu Gly Ala Ile Leu Asn Asn Leu Le - #u Pro Asn Met Ser Tyr
370 - # 375 - # 380
- - Val Leu Gln Ile Val Ala Ile Cys Thr Asn Gl - #y Leu Tyr Gly Lys Tyr
385 3 - #90 3 - #95 4 -
#00
- - Ser Asp Gln Leu Ile Val Asp Met Pro Thr As - #p Asn Pro Glu Leu
Asp
405 - # 410 - # 415
- - Leu Phe Pro Glu Leu Ile Gly Thr Glu Glu Il - #e Ile Lys Glu Glu Glu
420 - # 425 - # 430
- - Glu Gly Lys Asp Ile Glu Glu Gly Ala Ile Va - #l Asn Pro Gly Arg Asp
435 - # 440 - # 445
- - Ser Ala Thr Asn Gln Ile Arg Lys Lys Glu Pr - #o Gln Ile Ser Thr Thr
450 - # 455 - # 460
- - Thr His Tyr Asn Arg Ile Gly Thr Lys Tyr As - #n Glu Ala Lys Thr Asn
465 4 - #70 4 - #75 4 -
#80
- - Arg Ser Pro Thr Arg Gly Ser Glu Phe Ser Gl - #y Lys Gly Asp Val
Pro
485 - # 490 - # 495
- - Asn Thr Ser Leu Asn Ser Thr Ser Gln Pro Va - #l Thr Lys Leu Ala Thr
500 - # 505 - # 510
- - Glu Lys Asp Ile Ser Leu Thr Ser Gln Thr Va - #l Thr Glu Leu Pro Pro
515 - # 520 - # 525
- - His Thr Val Glu Gly Thr Ser Ala Ser Leu As - #n Asp Gly Ser Lys Thr
530 - # 535 - # 540
- - Val Leu Arg Ser Pro His Met Asn Leu Ser Gl - #y Thr Ala Glu Ser Leu
545 5 - #50 5 - #55 5 -
#60
- - Asn Thr Val Ser Ile Thr Glu Tyr Glu Glu Gl - #u Ser Leu Leu Thr
Ser
565 - # 570 - # 575
- - Phe Lys Leu Asp Thr Gly Ala Glu Asp Ser Se - #r Gly Ser Ser Pro Ala
580 - # 585 - # 590
- - Thr Ser Ala Ile Pro Phe Ile Ser Glu Asn Il - #e Ser Gln Gly Tyr Ile
595 - # 600 - # 605
- - Phe Ser Ser Glu Asn Pro Glu Thr Ile Thr Ty - #r Asp Val Leu Ile Pro
610 - # 615 - # 620
- - Glu Ser Ala Arg Asn Ala Ser Glu Asp Ser Th - #r Ser Ser Gly Ser Glu
625 6 - #30 6 - #35 6 -
#40
- - Glu Ser Leu Lys Asp Pro Ser Met Glu Gly As - #n Val Trp Phe Pro
Ser
645 - # 650 - # 655
- - Ser Thr Asp Ile Thr Ala Gln Pro Asp Val Gl - #y Ser Gly Arg Glu Ser
660 - # 665 - # 670
- - Phe Leu Gln Thr Asn Tyr Thr Glu Ile Arg Va - #l Asp Glu Ser Glu Lys
675 - # 680 - # 685
- - Thr Thr Lys Ser Phe Ser Ala Gly Pro Val Me - #t Ser Gln Gly Pro Ser
690 - # 695 - # 700
- - Val Thr Asp Leu Glu Met Pro His Tyr Ser Th - #r Phe Ala Tyr Phe Pro
705 7 - #10 7 - #15 7 -
#20
- - Thr Glu Val Thr Pro His Ala Phe Thr Pro Se - #r Ser Arg Gln Gln
Asp
725 - # 730 - # 735
- - Leu Val Ser Thr Val Asn Val Val Tyr Ser Gl - #n Thr Thr Gln Pro Val
740 - # 745 - # 750
- - Tyr Asn Gly Glu Thr Pro Leu Gln Pro Ser Ty - #r Ser Ser Glu Val Phe
755 - # 760 - # 765
- - Pro Leu Val Thr Pro Leu Leu Leu Asp Asn Gl - #n Ile Leu Asn Thr Thr
770 - # 775 - # 780
- - Pro Ala Ala Ser Ser Ser Asp Ser Ala Leu Hi - #s Ala Thr Pro Val Phe
785 7 - #90 7 - #95 8 -
#00
- - Pro Ser Val Asp Val Ser Phe Glu Ser Ile Le - #u Ser Ser Tyr Asp
Gly
805 - # 810 - # 815
- - Ala Pro Leu Leu Pro Phe Ser Ser Ala Ser Ph - #e Ser Ser Glu Leu Phe
820 - # 825 - # 830
- - Arg His Leu His Thr Val Ser Gln Ile Leu Pr - #o Gln Val Thr Ser Ala
835 - # 840 - # 845
- - Thr Glu Ser Asp Lys Val Pro Leu His Ala Se - #r Leu Pro Val Ala Gly
850 - # 855 - # 860
- - Gly Asp Leu Leu Leu Glu Pro Ser Leu Ala Gl - #n Tyr Ser Asp Val Leu
865 8 - #70 8 - #75 8 -
#80
- - Ser Thr Thr His Ala Ala Ser Lys Thr Leu Gl - #u Phe Gly Ser Glu
Ser
885 - # 890 - # 895
- - Gly Val Leu Tyr Lys Thr Leu Met Phe Ser Gl - #n Val Glu Pro Pro Ser
900 - # 905 - # 910
- - Ser Asp Ala Met Met His Ala Arg Ser Ser Gl - #y Pro Glu Pro Ser Tyr
915 - # 920 - # 925
- - Ala Leu Ser Asp Asn Glu Gly Ser Gln His Il - #e Phe Thr Val Ser Tyr
930 - # 935 - # 940
- - Ser Ser Ala Ile Pro Val His Asp Ser Val Gl - #y Val Thr Tyr Gln Gly
945 9 - #50 9 - #55 9 -
#60
- - Ser Leu Phe Ser Gly Pro Ser His Ile Pro Il - #e Pro Lys Ser Ser
Leu
965 - # 970 - # 975
- - Ile Thr Pro Thr Ala Ser Leu Leu Gln Pro Th - #r His Ala Leu Ser Gly
980 - # 985 - # 990
- - Asp Gly Glu Trp Ser Gly Ala Ser Ser Asp Se - #r Glu Phe Leu Leu Pro
995 - # 1000 - # 1005
- - Asp Thr Asp Gly Leu Thr Ala Leu Asn Ile Se - #r Ser Pro Val Ser Val
1010 - # 1015 - # 1020
- - Ala Glu Phe Thr Tyr Thr Thr Ser Val Phe Gl - #y Asp Asp Asn Lys Ala
1025 1030 - # 1035 - # 1040
- - Leu Ser Lys Ser Glu Ile Ile Tyr Gly Asn Gl - #u Thr Glu Leu Gln Ile
1045 - # 1050 - # 1055
- - Pro Ser Phe Asn Glu Met Val Tyr Pro Ser Gl - #u Ser Thr Val Met Pro
1060 - # 1065 - # 1070
- - Asn Met Tyr Asp Asn Val Asn Lys Leu Asn Al - #a Ser Leu Gln Glu Thr
1075 - # 1080 - # 1085
- - Ser Val Ser Ile Ser Ser Thr Lys Gly Met Ph - #e Pro Gly Ser Leu Ala
1090 - # 1095 - # 1100
- - His Thr Thr Thr Lys Val Phe Asp His Glu Il - #e Ser Gln Val Pro Glu
1105 1110 - # 1115 - # 1120
- - Asn Asn Phe Ser Val Gln Pro Thr His Thr Va - #l Ser Gln Ala Ser Gly
1125 - # 1130 - # 1135
- - Asp Thr Ser Leu Lys Pro Val Leu Ser Ala As - #n Ser Glu Pro Ala Ser
1140 - # 1145 - # 1150
- - Ser Asp Pro Ala Ser Ser Glu Met Leu Ser Pr - #o Ser Thr Gln Leu Leu
1155 - # 1160 - # 1165
- - Phe Tyr Glu Thr Ser Ala Ser Phe Ser Thr Gl - #u Val Leu Leu Gln Pro
1170 - # 1175 - # 1180
- - Ser Phe Gln Ala Ser Asp Val Asp Thr Leu Le - #u Lys Thr Val Leu Pro
1185 1190 - # 1195 - # 1200
- - Ala Val Pro Ser Asp Pro Ile Leu Val Glu Th - #r Pro Lys Val Asp Lys
1205 - # 1210 - # 1215
- - Ile Ser Ser Thr Met Leu His Leu Ile Val Se - #r Asn Ser Ala Ser Ser
1220 - # 1225 - # 1230
- - Glu Asn Met Leu His Ser Thr Ser Val Pro Va - #l Phe Asp Val Ser Pro
1235 - # 1240 - # 1245
- - Thr Ser His Met His Ser Ala Ser Leu Gln Gl - #y Leu Thr Ile Ser Tyr
1250 - # 1255 - # 1260
- - Ala Ser Glu Lys Tyr Glu Pro Val Leu Leu Ly - #s Ser Glu Ser Ser His
1265 1270 - # 1275 - # 1280
- - Gln Val Val Pro Ser Leu Tyr Ser Asn Asp Gl - #u Leu Phe Gln Thr Ala
1285 - # 1290 - # 1295
- - Asn Leu Glu Ile Asn Gln Ala His Pro Pro Ly - #s Gly Arg His Val Phe
1300 - # 1305 - # 1310
- - Ala Thr Pro Val Leu Ser Ile Asp Glu Pro Le - #u Asn Thr Leu Ile Asn
1315 - # 1320 - # 1325
- - Lys Leu Ile His Ser Asp Glu Ile Leu Thr Se - #r Thr Lys Ser Ser Val
1330 - # 1335 - # 1340
- - Thr Gly Lys Val Phe Ala Gly Ile Pro Thr Va - #l Ala Ser Asp Thr Phe
1345 1350 - # 1355 - # 1360
- - Val Ser Thr Asp His Ser Val Pro Ile Gly As - #n Gly His Val Ala Ile
1365 - # 1370 - # 1375
- - Thr Ala Val Ser Pro His Arg Asp Gly Ser Va - #l Thr Ser Thr Lys Leu
1380 - # 1385 - # 1390
- - Leu Phe Pro Ser Lys Ala Thr Ser Glu Leu Se - #r His Ser Ala Lys Ser
1395 - # 1400 - # 1405
- - Asp Ala Gly Leu Val Gly Gly Gly Glu Asp Gl - #y Asp Thr Asp Asp Asp
1410 - # 1415 - # 1420
- - Gly Asp Asp Asp Asp Asp Asp Arg Gly Ser As - #p Gly Leu Ser Ile His
1425 1430 - # 1435 - # 1440
- - Lys Cys Met Ser Cys Ser Ser Tyr Arg Glu Se - #r Gln Glu Lys Val Met
1445 - # 1450 - # 1455
- - Asn Asp Ser Asp Thr His Glu Asn Ser Leu Me - #t Asp Gln Asn Asn Pro
1460 - # 1465 - # 1470
- - Ile Ser Tyr Ser Leu Ser Glu Asn Ser Glu Gl - #u Asp Asn Arg Val Thr
1475 - # 1480 - # 1485
- - Ser Val Ser Ser Asp Ser Gln Thr Gly Met As - #p Arg Ser Pro Gly Lys
1490 - # 1495 - # 1500
- - Ser Pro Ser Ala Asn Gly Leu Ser Gln Lys Hi - #s Asn Asp Gly Lys Glu
1505 1510 - # 1515 - # 1520
- - Glu Asn Asp Ile Gln Thr Gly Ser Ala Leu Le - #u Pro Leu Ser Pro Glu
1525 - # 1530 - # 1535
- - Ser Lys Ala Trp Ala Val Leu Thr Ser Asp Gl - #u Glu Ser Gly Ser Gly
1540 - # 1545 - # 1550
- - Gln Gly Thr Ser Asp Ser Leu Asn Glu Asn Gl - #u Thr Ser Thr Asp Phe
1555 - # 1560 - # 1565
- - Ser Phe Ala Asp Thr Asn Glu Lys Asp Ala As - #p Gly Ile Leu Ala Ala
1570 - # 1575 - # 1580
- - Gly Asp Ser Glu Ile Thr Pro Gly Phe Pro Gl - #n Ser Pro Thr Ser Ser
1585 1590 - # 1595 - # 1600
- - Val Thr Ser Glu Asn Ser Glu Val Phe His Va - #l Ser Glu Ala Glu Ala
1605 - # 1610 - # 1615
- - Ser Asn Ser Ser His Glu Ser Arg Ile Gly Le - #u Ala Glu Gly Leu Glu
1620 - # 1625 - # 1630
- - Ser Glu Lys Lys Ala Val Ile Pro Leu Val Il - #e Val Ser Ala Leu Thr
1635 - # 1640 - # 1645
- - Phe Ile Cys Leu Val Val Leu Val Gly Ile Le - #u Ile Tyr Trp Arg Lys
1650 - # 1655 - # 1660
- - Cys Phe Gln Thr Ala His Phe Tyr Leu Glu As - #p Ser Thr Ser Pro Arg
1665 1670 - # 1675 - # 1680
- - Val Ile Ser Thr Pro Pro Thr Pro Ile Phe Pr - #o Ile Ser Asp Asp Val
1685 - # 1690 - # 1695
- - Gly Ala Ile Pro Ile Lys His Phe Pro Lys Hi - #s Val Ala Asp Leu His
1700 - # 1705 - # 1710
- - Ala Ser Ser Gly Phe Thr Glu Glu Phe Glu Gl - #u Val Gln Ser Cys Thr
1715 - # 1720 - # 1725
- - Val Asp Leu Gly Ile Thr Ala Asp Ser Ser As - #n His Pro Asp Asn Lys
1730 - # 1735 - # 1740
- - His Lys Asn Arg Tyr Ile Asn Ile Val Ala Ty - #r Asp His Ser Arg Val
1745 1750 - # 1755 - # 1760
- - Lys Leu Ala Gln Leu Ala Glu Lys Asp Gly Ly - #s Leu Thr Asp Tyr Ile
1765 - # 1770 - # 1775
- - Asn Ala Asn Tyr Val Asp Gly Tyr Asn Arg Pr - #o Lys Ala Tyr Ile Ala
1780 - # 1785 - # 1790
- - Ala Gln Gly Pro Leu Lys Ser Thr Ala Glu As - #p Phe Trp Arg Met Ile
1795 - # 1800 - # 1805
- - Trp Glu His Asn Val Glu Val Ile Val Met Il - #e Thr Asn Leu Val Glu
1810 - # 1815 - # 1820
- - Lys Gly Arg Arg Lys Cys Asp Gln Tyr Trp Pr - #o Ala Asp Gly Ser Glu
1825 1830 - # 1835 - # 1840
- - Glu Tyr Gly Asn Phe Leu Val Thr Gln Lys Se - #r Val Gln Val Leu Ala
1845 - # 1850 - # 1855
- - Tyr Tyr Thr Val Arg Asn Phe Thr Leu Arg As - #n Thr Lys Ile Lys Lys
1860 - # 1865 - # 1870
- - Gly Ser Gln Lys Gly Arg Pro Ser Gly Arg Va - #l Val Thr Gln Tyr His
1875 - # 1880 - # 1885
- - Tyr Thr Gln Trp Pro Asp Met Gly Val Pro Gl - #u Tyr Ser Leu Pro Val
1890 - # 1895 - # 1900
- - Leu Thr Phe Val Arg Lys Ala Ala Tyr Ala Ly - #s Arg His Ala Val Gly
1905 1910 - # 1915 - # 1920
- - Pro Val Val Val His Cys Ser Ala Gly Val Gl - #y Arg Thr Gly Thr Tyr
1925 - # 1930 - # 1935
- - Ile Val Leu Asp Ser Met Leu Gln Gln Ile Gl - #n His Glu Gly Thr Val
1940 - # 1945 - # 1950
- - Asn Ile Phe Gly Phe Leu Lys His Ile Arg Se - #r Gln Arg Asn Tyr Leu
1955 - # 1960 - # 1965
- - Val Gln Thr Glu Glu Gln Tyr Val Phe Ile Hi - #s Asp Thr Leu Val Glu
1970 - # 1975 - # 1980
- - Ala Ile Leu Ser Lys Glu Thr Glu Val Leu As - #p Ser His Ile His Ala
1985 1990 - # 1995 - # 2000
- - Tyr Val Asn Ala Leu Leu Ile Pro Gly Pro Al - #a Gly Lys Thr Lys Leu
2005 - # 2010 - # 2015
- - Glu Lys Gln Phe Gln Leu Leu Ser Gln Ser As - #n Ile Gln Gln Ser Asp
2020 - # 2025 - # 2030
- - Tyr Ser Ala Ala Leu Lys Gln Cys Asn Arg Gl - #u Lys Asn Arg Thr Ser
2035 - # 2040 - # 2045
- - Ser Ile Ile Pro Val Glu Arg Ser Arg Val Gl - #y Ile Ser Ser Leu Ser
2050 - # 2055 - # 2060
- - Gly Glu Gly Thr Asp Tyr Ile Asn Ala Ser Ty - #r Ile Met Gly Tyr Tyr
2065 2070 - # 2075 - # 2080
- - Gln Ser Asn Glu Phe Ile Ile Thr Gln His Pr - #o Leu Leu His Thr Ile
2085 - # 2090 - # 2095
- - Lys Asp Phe Trp Arg Met Ile Trp Asp His As - #n Ala Gln Leu Val Val
2100 - # 2105 - # 2110
- - Met Ile Pro Asp Gly Gln Asn Met Ala Glu As - #p Glu Phe Val Tyr Trp
2115 - # 2120 - # 2125
- - Pro Asn Lys Asp Glu Pro Ile Asn Cys Glu Se - #r Phe Lys Val Thr Leu
2130 - # 2135 - # 2140
- - Met Ala Glu Glu His Lys Cys Leu Ser Asn Gl - #u Glu Lys Leu Ile Ile
2145 2150 - # 2155 - # 2160
- - Gln Asp Phe Ile Leu Glu Ala Thr Gln Asp As - #p Tyr Val Leu Glu Val
2165 - # 2170 - # 2175
- - Arg His Phe Gln Cys Pro Lys Trp Pro Asn Pr - #o Asp Ser Pro Ile Ser
2180 - # 2185 - # 2190
- - Lys Thr Phe Glu Leu Ile Ser Val Ile Lys Gl - #u Glu Ala Ala Asn Arg
2195 - # 2200 - # 2205
- - Asp Gly Pro Met Ile Val His Asp Glu His Gl - #y Gly Val Thr Ala Gly
2210 - # 2215 - # 2220
- - Thr Phe Cys Ala Leu Thr Thr Leu Met His Gl - #n Leu Glu Lys Glu Asn
2225 2230 - # 2235 - # 2240
- - Ser Val Asp Val Tyr Gln Val Ala Lys Met Il - #e Asn Leu Met Arg Pro
2245 - # 2250 - # 2255
- - Gly Val Phe Ala Asp Ile Glu Gln Tyr Gln Ph - #e Leu Tyr Lys Val Ile
2260 - # 2265 - # 2270
- - Leu Ser Leu Val Ser Thr Arg Gln Glu Glu As - #n Pro Ser Thr Ser Leu
2275 - # 2280 - # 2285
- - Asp Ser Asn Gly Ala Ala Leu Pro Asp Gly As - #n Ile Ala Glu Ser Leu
2290 - # 2295 - # 2300
- - Glu Ser Leu Val
2305
- - - - (2) INFORMATION FOR SEQ ID NO:3:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 267 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
- - Gly Asp Pro Tyr Trp Ala Tyr Ser Gly Ala Ty - #r Gly Pro Glu His Trp
1 5 - # 10 - # 15
- - Val Thr Ser Ser Val Ser Cys Gly Gly Arg Hi - #s Gln Ser Pro Ile Asp
20 - # 25 - # 30
- - Ile Leu Asp Gln Tyr Ala Arg Val Gly Glu Gl - #u Tyr Gln Glu Leu Gln
35 - # 40 - # 45
- - Leu Asp Gly Phe Asp Asn Glu Ser Ser Asn Ly - #s Thr Trp Met Lys Asn
50 - # 55 - # 60
- - Thr Gly Lys Thr Val Ala Ile Leu Leu Lys As - #p Asp Tyr Phe Val Ser
65 - #70 - #75 - #80
- - Gly Ala Gly Leu Pro Gly Arg Phe Lys Ala Gl - #u Lys Val Glu Phe His
85 - # 90 - # 95
- - Trp Gly His Ser Asn Gly Ser Ala Gly Ser Gl - #u His Ser Ile Asn Gly
100 - # 105 - # 110
- - Arg Arg Phe Pro Val Glu Met Gln Ile Phe Ph - #e Tyr Asn Pro Asp Asp
115 - # 120 - # 125
- - Phe Asp Ser Phe Gln Thr Ala Ile Ser Glu As - #n Arg Ile Ile Gly Ala
130 - # 135 - # 140
- - Met Ala Ile Phe Phe Gln Val Ser Pro Arg As - #p Asn Ser Ala Leu Asp
145 1 - #50 1 - #55 1 -
#60
- - Pro Ile Ile His Gly Leu Lys Gly Val Val Hi - #s His Glu Lys Glu
Thr
165 - # 170 - # 175
- - Phe Leu Asp Pro Phe Val Leu Arg Asp Leu Le - #u Pro Ala Ser Leu Gly
180 - # 185 - # 190
- - Ser Tyr Tyr Arg Tyr Thr Gly Ser Leu Thr Th - #r Pro Pro Cys Ser Glu
195 - # 200 - # 205
- - Ile Val Glu Trp Ile Val Phe Arg Arg Pro Va - #l Pro Ile Ser Tyr His
210 - # 215 - # 220
- - Gln Leu Glu Ala Phe Tyr Ser Ile Phe Thr Th - #r Glu Gln Gln Asp His
225 2 - #30 2 - #35 2 -
#40
- - Val Lys Ser Val Glu Tyr Leu Arg Asn Asn Ph - #e Arg Pro Gln Gln
Arg
245 - # 250 - # 255
- - Leu His Asp Arg Val Val Ser Lys Ser Ala Va - #l
260 - # 265
- - - - (2) INFORMATION FOR SEQ ID NO:4:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
- - Ala Ser Pro Asp Trp Gly Tyr Asp Asp Lys As - #n Gly Pro Glu Gln Trp
1 5 - # 10 - # 15
- - Ser Lys Leu Tyr Pro Ile Ala Asn Gly Asn As - #n Gln Ser Pro Val Asp
20 - # 25 - # 30
- - Ile Lys Thr Ser Glu Thr Lys His Asp Thr Se - #r Leu Lys Pro Ile Ser
35 - # 40 - # 45
- - Val Ser Tyr Asn Pro Ala Thr Ala Lys Glu Il - #e Ile Asn Val Gly His
50 - # 55 - # 60
- - Ser Phe His Val Asn Phe Glu Asp Asn Asp As - #n Arg Ser Val Leu Lys
65 - #70 - #75 - #80
- - Gly Gly Pro Phe Ser Asp Ser Tyr Arg Leu Ph - #e Gln Phe His Phe His
85 - # 90 - # 95
- - Trp Gly Ser Thr Asn Glu His Gly Ser Glu Hi - #s Thr Val Asp Gly Val
100 - # 105 - # 110
- - Lys Tyr Ser Ala Glu Leu His Val Ala His Tr - #p Asn Ser Ala Lys Tyr
115 - # 120 - # 125
- - Ser Ser Leu Ala Glu Ala Ala Ser Lys Ala As - #p Gly Leu Ala Val Ile
130 - # 135 - # 140
- - Gly Val Leu Met Lys Val Gly Glu Ala Asn Pr - #o Lys Leu Gln Lys Val
145 1 - #50 1 - #55 1 -
#60
- - Leu Asp Ala Leu Gln Ala Ile Lys Thr Lys Gl - #y Lys Arg Ala Pro
Phe
165 - # 170 - # 175
- - Thr Asn Phe Asp Pro Ser Thr Leu Leu Pro Se - #r Ser Leu Asp Phe Trp
180 - # 185 - # 190
- - Thr Tyr Pro Gly Ser Leu Thr His Pro Pro Le - #u Tyr Glu Ser Val Thr
195 - # 200 - # 205
- - Trp Ile Ile Cys Lys Glu Ser Ile Ser Val Se - #r Ser Glu Gln Leu Ala
210 - # 215 - # 220
- - Gln Phe Arg Ser Leu Leu Ser Asn Val Glu Gl - #y Asp Asn Ala Val Pro
225 2 - #30 2 - #35 2 -
#40
- - Met Gln His Asn Asn Arg Pro Thr Gln Pro Le - #u Lys Gly Arg Thr
Val
245 - # 250 - # 255
- - Arg Ala Ser Phe
260
- - - - (2) INFORMATION FOR SEQ ID NO:5:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 259 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
- - Ser His His Trp Gly Tyr Gly Lys His Asn Gl - #y Pro Glu His Trp His
1 5 - # 10 - # 15
- - Lys Asp Phe Pro Ile Ala Lys Gly Glu Arg Gl - #n Ser Pro Val Asp Ile
20 - # 25 - # 30
- - Asp Thr His Thr Ala Lys Tyr Asp Pro Ser Le - #u Lys Pro Leu Ser Val
35 - # 40 - # 45
- - Ser Tyr Asp Gln Ala Thr Ser Leu Arg Ile Le - #u Asn Asn Gly His Ala
50 - # 55 - # 60
- - Phe Asn Val Glu Phe Asp Asp Ser Gln Asp Ly - #s Ala Val Leu Lys Gly
65 - #70 - #75 - #80
- - Gly Pro Leu Asp Gly Thr Tyr Arg Leu Ile Gl - #n Phe His Phe His Trp
85 - # 90 - # 95
- - Gly Ser Leu Asp Gly Gln Gly Ser Glu His Th - #r Val Asp Lys Lys Lys
100 - # 105 - # 110
- - Tyr Ala Ala Glu Leu His Leu Val His Trp As - #n Thr Lys Tyr Gly Asp
115 - # 120 - # 125
- - Phe Gly Lys Ala Val Gln Gln Pro Asp Gly Le - #u Ala Val Leu Gly Ile
130 - # 135 - # 140
- - Phe Leu Lys Val Gly Ser Ala Lys Pro Gly Le - #u Gln Lys Val Val Asp
145 1 - #50 1 - #55 1 -
#60
- - Val Leu Asp Ser Ile Lys Thr Lys Gly Lys Se - #r Ala Asp Phe Thr
Asn
165 - # 170 - # 175
- - Phe Asp Pro Arg Gly Leu Leu Pro Glu Ser Le - #u Asp Tyr Trp Thr Tyr
180 - # 185 - # 190
- - Pro Gly Ser Leu Thr Thr Pro Pro Leu Leu Gl - #u Cys Val Thr Trp Ile
195 - # 200 - # 205
- - Val Leu Lys Glu Pro Ile Ser Val Ser Ser Gl - #u Gln Val Leu Lys Phe
210 - # 215 - # 220
- - Arg Lys Leu Asn Phe Asn Gly Glu Gly Glu Pr - #o Glu Glu Leu Met Val
225 2 - #30 2 - #35 2 -
#40
- - Asp Asn Trp Arg Pro Ala Gln Pro Leu Lys As - #n Arg Gln Ile Lys
Ala
245 - # 250 - # 255
- - Ser Phe Lys
- - - - (2) INFORMATION FOR SEQ ID NO:6:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 259 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
- - Ala Lys Glu Trp Gly Tyr Ala Ser His Asn Gl - #y Pro Asp His Trp His
1 5 - # 10 - # 15
- - Glu Leu Phe Pro Asn Ala Lys Gly Glu Asn Gl - #n Ser Pro Ile Glu Leu
20 - # 25 - # 30
- - His Thr Lys Asp Ile Arg His Asp Pro Ser Le - #u Gln Pro Trp Ser Val
35 - # 40 - # 45
- - Ser Tyr Asp Gly Gly Ser Ala Lys Thr Ile Le - #u Asn Asn Gly Lys Thr
50 - # 55 - # 60
- - Cys Arg Val Val Phe Asp Asp Thr Tyr Asp Ar - #g Ser Met Leu Arg Gly
65 - #70 - #75 - #80
- - Gly Pro Leu Pro Gly Pro Tyr Arg Leu Arg Gl - #n Phe His Leu His Trp
85 - # 90 - # 95
- - Gly Ser Ser Asp Asp His Gly Ser Glu His Th - #r Val Asp Gly Val Lys
100 - # 105 - # 110
- - Tyr Ala Ala Glu Leu His Leu Val His Trp As - #n Pro Lys Tyr Asn Thr
115 - # 120 - # 125
- - Phe Lys Glu Ala Leu Lys Gln Arg Asp Gly Il - #e Ala Val Ile Gly Ile
130 - # 135 - # 140
- - Phe Leu Lys Ile Gly His Glu Asn Gly Glu Ph - #e Gln Ile Phe Leu Asp
145 1 - #50 1 - #55 1 -
#60
- - Ala Leu Asp Lys Ile Lys Thr Lys Gly Lys Gl - #u Ala Pro Phe Thr
Lys
165 - # 170 - # 175
- - Phe Asp Pro Ser Cys Leu Phe Pro Ala Cys Ar - #g Asp Tyr Trp Thr Tyr
180 - # 185 - # 190
- - Gln Gly Ser Phe Thr Thr Pro Pro Cys Glu Gl - #u Cys Ile Val Trp Leu
195 - # 200 - # 205
- - Leu Leu Lys Glu Pro Met Thr Val Ser Ser As - #p Gln Met Ala Lys Leu
210 - # 215 - # 220
- - Arg Ser Leu Leu Ser Ser Ala Glu Asn Glu Pr - #o Pro Val Pro Leu Val
225 2 - #30 2 - #35 2 -
#40
- - Ser Asn Trp Arg Pro Pro Gln Pro Ile Asn As - #n Arg Val Val Arg
Ala
245 - # 250 - # 255
- - Ser Phe Lys
- - - - (2) INFORMATION FOR SEQ ID NO:7:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
- - Ala Glu Ser His Trp Cys Tyr Glu Val Gln Al - #a Glu Ser Ser Asn Tyr
1 5 - # 10 - # 15
- - Pro Cys Leu Val Pro Val Lys Trp Gly Gly As - #n Cys Gln Lys Asp Arg
20 - # 25 - # 30
- - Gln Ser Pro Ile Asn Ile Val Thr Thr Lys Al - #a Lys Val Asp Lys Lys
35 - # 40 - # 45
- - Leu Gly Arg Phe Phe Phe Ser Gly Tyr Asp Ly - #s Lys Gln Thr Trp Thr
50 - # 55 - # 60
- - Val Gln Asn Asn Gly His Ser Val Met Met Le - #u Leu Glu Asn Lys Ala
65 - #70 - #75 - #80
- - Ser Ile Ser Gly Gly Gly Leu Pro Ala Pro Ty - #r Gln Ala Lys Gln Leu
85 - # 90 - # 95
- - His Leu His Trp Ser Asp Leu Pro Tyr Lys Gl - #y Ser Glu His Ser Leu
100 - # 105 - # 110
- - Asp Gly Glu His Phe Ala Met Glu Met His Il - #e Val His Glu Lys Glu
115 - # 120 - # 125
- - Lys Gly Thr Ser Arg Asn Val Lys Glu Ala Gl - #n Asp Pro Glu Asp Glu
130 - # 135 - # 140
- - Ile Ala Val Leu Ala Phe Leu Val Glu Ala Gl - #y Thr Gln Val Asn Glu
145 1 - #50 1 - #55 1 -
#60
- - Gly Phe Gln Pro Leu Val Glu Ala Leu Ser As - #n Ile Pro Lys Pro
Glu
165 - # 170 - # 175
- - Met Ser Thr Thr Met Ala Glu Ser Ser Leu Le - #u Asp Leu Leu Pro Lys
180 - # 185 - # 190
- - Glu Glu Lys Leu Arg His Tyr Phe Arg Tyr Le - #u Gly Ser Leu Thr Thr
195 - # 200 - # 205
- - Pro Thr Cys Asp Glu Lys Val Val Trp Thr Va - #l Phe Arg Glu Pro Ile
210 - # 215 - # 220
- - Gln Leu His Arg Glu Gln Ile Leu Ala Phe Se - #r Gln Lys Leu Tyr Tyr
225 2 - #30 2 - #35 2 -
#40
- - Asp Lys Glu Gln Thr Val Ser Met Lys Asp As - #n Val Arg Pro Leu
Gln
245 - # 250 - # 255
- - Gln Leu Gly Gln Arg Thr Val Ile Lys Ser Gl - #y Ala
260 - # 265
- - - - (2) INFORMATION FOR SEQ ID NO:8:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
- - Gln His Val Ser Asp Trp Thr Tyr Ser Glu Gl - #y Ala Leu Asp Glu Ala
1 5 - # 10 - # 15
- - His Trp Pro Gln His Tyr Pro Ala Cys Gly Gl - #y Gln Arg Gln Ser Pro
20 - # 25 - # 30
- - Ile Asn Leu Gln Arg Thr Lys Val Arg Tyr As - #n Pro Ser Leu Lys Gly
35 - # 40 - # 45
- - Leu Asn Met Thr Gly Tyr Glu Thr Gln Ala Gl - #y Glu Phe Pro Met Val
50 - # 55 - # 60
- - Asn Asn Gly His Thr Val Gln Ile Gly Leu Pr - #o Ser Thr Met Arg Met
65 - #70 - #75 - #80
- - Thr Val Ala Asp Gly Ile Val Tyr Ile Ala Gl - #n Gln Met His Phe His
85 - # 90 - # 95
- - Trp Gly Gly Ala Ser Ser Glu Ile Ser Gly Se - #r Glu His Thr Val Asp
100 - # 105 - # 110
- - Gly Ile Arg His Val Ile Glu Ile His Ile Va - #l His Tyr Asn Ser Lys
115 - # 120 - # 125
- - Tyr Lys Thr Tyr Asp Ile Ala Gln Asp Ala Pr - #o Asp Gly Leu Ala Val
130 - # 135 - # 140
- - Leu Ala Ala Phe Val Glu Val Lys Asn Tyr Pr - #o Glu Asn Thr Tyr Tyr
145 1 - #50 1 - #55 1 -
#60
- - Ser Asn Phe Ile Ser His Leu Ala Asn Ile Ly - #s Tyr Pro Gly Gln
Arg
165 - # 170 - # 175
- - Thr Thr Leu Thr Gly Leu Asp Val Gln Asp Me - #t Leu Pro Arg Asn Leu
180 - # 185 - # 190
- - Gln His Tyr Tyr Thr Tyr His Gly Ser Leu Th - #r Thr Pro Pro Cys Thr
195 - # 200 - # 205
- - Glu Asn Val His Trp Phe Val Leu Ala Asp Ph - #e Val Lys Leu Ser Arg
210 - # 215 - # 220
- - Thr Gln Val Trp Lys Leu Glu Asn Ser Leu Le - #u Asp His Arg Asn Lys
225 2 - #30 2 - #35 2 -
#40
- - Thr Ile His Asn Asp Tyr Arg Arg Thr Gln Pr - #o Leu Asn His Arg
Val
245 - # 250 - # 255
- - Val Glu Ser Asn Phe Pro
260
- - - - (2) INFORMATION FOR SEQ ID NO:9:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 261 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
- - Gly His His Gly Trp Gly Tyr Gly Gln Asp As - #p Gly Pro Ala Ser His
1 5 - # 10 - # 15
- - Trp His Lys Leu Tyr Pro Ile Ala Gln Gly As - #p Arg Gln Ser Pro Ile
20 - # 25 - # 30
- - Asn Ile Ile Ser Ser Gln Ala Val Tyr Ser Pr - #o Ser Leu Gln Pro Leu
35 - # 40 - # 45
- - Glu Leu Ser Tyr Glu Ala Cys Met Ser Leu Se - #r Ile Thr Asn Asn Gly
50 - # 55 - # 60
- - His Ser Val Gln Val Asp Phe Asn Asp Ser As - #p Asp Arg Thr Val Val
65 - #70 - #75 - #80
- - Thr Gly Gly Pro Leu Glu Gly Pro Tyr Arg Le - #u Lys Gln Phe His Phe
85 - # 90 - # 95
- - His Trp Gly Lys Lys His Asp Val Gly Ser Gl - #u His Thr Val Asp Gly
100 - # 105 - # 110
- - Lys Ser Phe Pro Ser Glu Leu His Leu Val Hi - #s Trp Asn Ala Lys Lys
115 - # 120 - # 125
- - Tyr Ser Thr Phe Gly Glu Ala Ala Ser Ala Pr - #o Asp Gly Leu Ala Val
130 - # 135 - # 140
- - Gly Val Phe Leu Glu Thr Gly Asp Glu His Pr - #o Ser Met Asn Arg Leu
145 1 - #50 1 - #55 1 -
#60
- - Thr Asp Ala Leu Tyr Met Val Arg Phe Lys Gl - #y Thr Lys Ala Gln
Phe
165 - # 170 - # 175
- - Ser Cys Phe Asn Pro Lys Cys Leu Leu Pro Al - #a Ser Arg His Tyr Trp
180 - # 185 - # 190
- - Thr Tyr Pro Gly Ser Leu Thr Thr Pro Pro Le - #u Ser Glu Ser Val Thr
195 - # 200 - # 205
- - Trp Ile Val Leu Arg Glu Pro Ile Cys Ile Se - #r Glu Arg Gln Met Gly
210 - # 215 - # 220
- - Lys Phe Arg Ser Leu Leu Phe Thr Ser Glu As - #p Asp Glu Arg Ile His
225 2 - #30 2 - #35 2 -
#40
- - Met Val Asn Asn Phe Arg Pro Pro Gln Pro Le - #u Lys Gly Arg Val
Val
245 - # 250 - # 255
- - Lys Ala Ser Phe Arg
260
- - - - (2) INFORMATION FOR SEQ ID NO:10:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
- - Lys Leu Val Glu Glu Met Gly Trp Ser Tyr Th - #r Gly Ala Leu Asn Gln
1 5 - # 10 - # 15
- - Lys Asn Trp Gly Lys Lys Tyr Pro Ile Cys As - #n Ser Pro Lys Gln Ser
20 - # 25 - # 30
- - Pro Ile Asn Ile Asp Glu Asp Leu Thr Gln Va - #l Asn Val Asn Leu Lys
35 - # 40 - # 45
- - Lys Leu Lys Phe Gln Gly Trp Glu Lys Pro Se - #r Leu Glu Asn Thr Phe
50 - # 55 - # 60
- - Ile His Asn Thr Gly Lys Thr Val Glu Ile As - #n Leu Thr Asn Asp Tyr
65 - #70 - #75 - #80
- - Tyr Leu Ser Gly Gly Leu Ser Glu Lys Val Ph - #e Lys Ala Ser Lys Met
85 - # 90 - # 95
- - Thr Phe His Trp Gly Lys Cys Asn Val Ser Se - #r Glu Gly Ser Glu His
100 - # 105 - # 110
- - Ser Leu Glu Gly Gln Lys Phe Pro Leu Glu Me - #t Gln Val Tyr Cys Phe
115 - # 120 - # 125
- - Asp Ala Asp Arg Phe Ser Ser Phe Glu Glu Th - #r Val Lys Gly Lys Gly
130 - # 135 - # 140
- - Arg Leu Arg Ala Leu Ser Ile Leu Phe Glu Il - #e Gly Val Glu Glu Asn
145 1 - #50 1 - #55 1 -
#60
- - Leu Asp Tyr Lys Ala Ile Ile Asp Gly Thr Gl - #u Ser Val Ser Arg
Phe
165 - # 170 - # 175
- - Gly Lys Gln Ala Ala Leu Asp Pro Phe Ile Le - #u Gln Asn Leu Leu Pro
180 - # 185 - # 190
- - Asn Ser Thr Asp Lys Tyr Tyr Ile Tyr Asn Gl - #y Ser Leu Thr Ser Pro
195 - # 200 - # 205
- - Pro Cys Thr Asp Thr Val Glu Trp Ile Val Ph - #e Lys Asp Thr Val Ser
210 - # 215 - # 220
- - Ile Ser Glu Ser Gln Leu Pro Val Phe Cys Gl - #u Val Leu Thr Met Gln
225 2 - #30 2 - #35 2 -
#40
- - Gln Ser Gly Tyr Val Met Leu Met Asp Tyr Le - #u Gln Asn Asn Phe
Arg
245 - # 250 - # 255
- - Glu Gln Gln Tyr
260
- - - - (2) INFORMATION FOR SEQ ID NO:11:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: peptide
- - (ix) FEATURE:
(A) NAME/KEY: Modified-sit - #e
(B) LOCATION: 3
(D) OTHER INFORMATION: - #/label= Xaa
/note= - #"Xaa = Any amino acid"
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
- - Ser Gly Xaa Gly
1
- - - - (2) INFORMATION FOR SEQ ID NO:12:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: peptide
- - (ix) FEATURE:
(A) NAME/KEY: Modified-sit - #e
(B) LOCATION: 2
(D) OTHER INFORMATION: - #/label= Xaa
/note= - #"Xaa = Any amino acid"
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
- - Asp Xaa Asn Asp Asn
1 5
- - - - (2) INFORMATION FOR SEQ ID NO:13:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
- - (ii) MOLECULE TYPE: peptide
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
- - Asp Val Asn Glu
__________________________________________________________________________
Top